Therapy for subarachnoid hemorrhage and ischemia

ABSTRACT

The application provides data from a clinical trial of a PSD-95 inhibitor in subjects undergoing endovascular repair of an aneurysm in or otherwise affecting the CNS. The subjects were stratified by whether the aneurysm ruptured before performing the endovascular surgery. Rupture is associated with higher mortality or increased debilitation if a subject survives. The trial provided evidence of significant benefit in subjects with and without aneurysm rupture before endovascular was surgery performed. Surprisingly, the subjects benefitting most from treatment as judged both by pathology and neurocognitive outcome were those in which the aneurysm had ruptured causing a subarachnoid hemorrhage. These data constitute evidence that a PSD-95 inhibitor is beneficial not only in ischemic and hemorrhagic stroke but in forms of hemorrhage in or affecting the CNS, particularly, subarachnoid hemorrhage.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application continuation of U.S. application Ser. No.14/965,694 filed Dec. 10, 2015 now U.S. Pat. No. 10,300,110, which is acontinuation of U.S. application Ser. No. 13/774,053 filed Feb. 22, 2013now U.S. Pat. No. 9,241,970, which is a continuation-in-part of U.S.application Ser. No. 13/713,489, filed Dec. 13, 2012 now abandoned andPCT/IB2012/057259 filed Dec. 13, 2012, both of which claim the benefitof US Provisional No. 61/617,001 filed Mar. 28, 2012, US Provisional No.61/570,264, filed Dec. 13, 2011 and CA 2762338, filed Dec. 15, 2011,each incorporated by reference in its entirety for all purposes.

SEQUENCE LISTING

This application includes a sequence listing submitted herewith as atext file named “530494_SEQLST.txt” created on Apr. 11, 2019, andcontaining 16,365 bytes. The material contained in this text file ishereby incorporated by reference in its entirety.

BACKGROUND

Each year approximately 800,000 individuals in the USA suffer a stroke,with yearly direct and indirect societal costs that exceed $40 B. Strokeranks third among all causes of death. Currently, only therapies thatinduce reperfusion of an ischemic brain are widely approved astreatments for acute stroke (e.g., thrombolysis with alteplase (tissueplasminogen activator or rt-PA). These balance improved overall outcomewith the potential for serious complications and are underused. Safepharmacological neuroprotection, brain salvage by enhancing the brain'sresilience to ischemia, could dramatically enhance the number ofpatients that could benefit from acute stroke treatment. However, overdecades, research has failed to translate over 1000 neuroprotectivetreatments from discovery in cells and rodents to utility in humans, andclinical trials of putative neuroprotectants have failed. Thisscientific crisis gave rise to a prevailing paradigm thatpharmacological neuroprotection is not feasible or practicable inhumans. Thus there is an urgent unmet need to determine whether or notneuroprotection in humans is possible.

Stroke can be the result of ischemia or hemorrhage. Hemorrhagic strokeaccounts for about 17% of strokes but gives rise to a disproportionateshare of deaths and debilitating injury. Hemorrhagic stroke isaggravated rather than alleviated by the only approved stroke drugs,such as tPA, which act to restore blood flow. Too often, the timerequired to bring a subject to a hospital, reach an initial diagnosisand perform a brain scan to distinguish between ischemic and hemorrhagicstroke would place a subject outside the window in which tPA can beeffective. Thus, many ischemic stroke subjects, who could benefit fromtPA, do not receive it.

Hemorrhage in or proximate to the CNS can also occur independently ofischemic stroke, particularly in subarachnoid hemorrhages, and dural orsubdural hematoma and brain contusions. Such hemorrhages can arise as aresult of physical trauma, such as a fall or other blow to the head orfrom shaken baby syndrome. Although the immediate symptoms of suchhemorrhages can range from deceptively mild to severe, they can allrapidly become severe and life threatening. Such hemorrhages are thus alife-threatening emergency that even with the best current treatmentoften results in death or debilitating injury.

Subarachnoid Hemorrhage (SAH) is characterized by bleeding into thesubarachnoid space. SAH is a serious, acute, life-threatening event thatcan result in chronic debilitation. In about 85% of cases of spontaneousSAH, the cause is the rupture of an intracranial aneurysm, termedaneurysmal SAH. Aneurysmal SAH most commonly affects people between theages of 40 and 60 years, and is more likely to occur in women. Theincidence of aneurysmal SAH is 10 in every 100,000 individuals per yearin the U.S. Other less common causes of SAH include conditions such asvascular malformations. Acquired risk factors include high bloodpressure, alcohol abuse, drug abuse, smoking, and contraceptive use.Other risk factors include aneurysm in other blood vessels,fibromuscular dysplasia and other connective tissue disorders, andhistory of polycystic kidney disease.

SAH is a multiphasic event, with an acute brain insult that occurs atthe time of the initial bleed which is followed by secondary potentiallyinjurious events such as ischemia that occur from cerebral vasospasm andhydrocephalus. In the acute SAH-induced injury, distribution of blood inthe subarachnoid space, elevation of intracranial pressure (ICP),reduced cerebral perfusion pressure (CPP) and cerebral blood flow (CBF)initiate an acute injury cascade that produces transient brain ischemia,brain trauma due to the impulse produced by the sudden rise in ICP and,in some cases, brain injury due to intracerebral hematoma formation.Additionally, these initial events may lead to direct microvascularinjury, plugging of vessels and release of vasoactive substances byplatelet aggregates.

Secondary ischemic processes include anaerobic cellular respiration,energy depletion, impaired protein synthesis, excitotoxicity, freeradical attack, neuronal stress, deoxyribonucleic acid (DNA) damage,apoptosis and necrosis, alterations in nitric oxide (NO)/nitric oxidesynthase (NOS) pathways and lipid peroxidation. Although there is broadagreement about the range of secondary processes that may participate inproducing brain injury following SAH, the precise contribution ofindividual mechanisms during the acute injury period remain incompletelyunderstood.

Cerebral ischemia in SAH is the result of cerebral arterial vasospasm,and complicates the clinical course of approximately 30% of cases. Theincidence of clinically-relevant vasospasm in SAH is highest betweendays 5 and 12 after the SAH. However, this complication is quiteuncommon in the first three days after a SAH. A patient's ultimateclinical outcome after a SAH likely depends on the several factors,including demographic factors such as age and co-morbidities, theseverity of the SAH, and the various complications of the SAH such ashydrocephalus and vasospasm. Thus, cerebral ischemia due to vasospasm isnot the sole contributor to an adverse clinical outcome from SAH assymptoms appear immediately after rupture.

A different form of treatment for stroke and related conditions is nowin clinical trials (see WO 2010144721 and Aarts et al., Science 298,846-850 (2002)). This treatment uses TAT-NR2B9C, also known asTat-NR2B9c (YGRKKRRQRRRKLSSIESDV; SEQ ID NO:6], an agent that inhibitsPSD-95 binding to NMDAR 2 family members, thus reducing excitotoxicityinduced by cerebral ischemia. Treatment has been reported to reduceinfarction size and functional deficits in ischemic stroke and traumaticbrain injuries.

SUMMARY OF THE CLAIMED INVENTION

The invention provides methods of treating a damaging effect ofsubarachnoid hemorrhage in a subject, comprising administering an agentthat inhibits binding of PSD-95 to an NMDAR2 subunit to a subject havinga subarachnoid hemorrhage. In some methods, the subarachnoid hemorrhageis the result of physical trauma. In some methods, the subarachnoidhemorrhage occurs spontaneously. In some methods, the subarachnoidhemorrhage is due to a ruptured aneurysm. In some methods, thesubarachnoid hemorrhage is due to arteriovenous malformation. In somemethods, the agent inhibits development of neurocognitive deficits inthe subject. In some methods, the agent inhibits development ofinfarctions detectable by MRI. In some methods, the subject receivesendovascular surgery to repair a leaking blood vessel causing thesubarachnoid hemorrhage. In some methods, the agent reduces painresulting from endovascular surgery. In some methods, the pain is alonga path traversed by an endoscope used in performing the endoscopicsurgery. In some methods, the agent is administered by day 4 postrupture causing the subarachnoid hemorrhage. In some methods, the agentis administered on multiple occasions within twelve days post rupturecausing the subarachnoid hemorrhage. In some methods, the agent isadministered twice daily for at least two days or once daily for atleast three days. In some methods, the dose of the agent is 1-3 mg/kg.

The invention further provides methods of treating a damaging effect ofintracerebral hemorrhage in a subject, comprising administering an agentthat inhibits binding of PSD-95 to NMDAR2 subunit to a subject having anintracerebral hemorrhage. In some methods, the intracerebral hemorrhageis due to high blood pressure. In some methods, the intracerebralhemorrhage is due to a drug. In some methods, the drug is ananti-coagulant. In some methods, the agent inhibits development ofneurocognitive deficits in the subject. In some methods, the agentinhibits development of infarcts in the CNS detectable by MRI. In somemethods, the agent is administered before, during or after surgery torepair a blood vessel causing the hemorrhage. In some methods, the agentis administered before, during or after administering another agenteffect to reduce damaging effects of intracerebral hemorrhage.

The invention further provides an agent that inhibits binding of PSD-95to an NMDAR2 subunit for use in reducing a damaging effect of ischemiaor hemorrhage in or otherwise affecting the CNS of a subject andreducing pain in the subject from surgery to remediate the ischemia orhemorrhage. In some methods, the surgery is endovascular surgery. Insome methods, the pain is along a path traversed by an endoscope used inperforming the endoscopic surgery.

The invention further provides a method of reducing pain, comprisingadministering an agent that inhibits binding of PSD-95 to an NMDAR2subunit to a subject receiving endovascular surgery wherein the agentreduces pain in the subject resulting from the endovascular surgery. Insome methods, the pain is along a path traversed by an endoscope used inperforming the endoscopic surgery.

The invention further provides methods of inhibiting a damaging effectof ischemia or hemorrhage in or otherwise affecting the CNS in a subjectcomprising administering to a subject having or suspect of havingischemia or hemorrhage in or otherwise affecting the CNS an effectiveregime of an agent that inhibits binding of PSD95 to an NMDAR2 subunit,wherein the subject has not been given a scan to distinguish ischemiafrom hemorrhage when the administering is performed. In some methods,the subject has a hemorrhage in or otherwise affecting the CNS. In somemethods, the subject is not subject to reperfusion therapy.

The invention further provides methods of treating a damaging effect ofischemia or hemorrhage on the central nervous system, comprisingadministering an agent that inhibits binding of PSD-95 to NMDAR2subunits to a subject having or at risk of ischemia or hemorrhage. Insome methods, the agent is administered in conjunction with reperfusiontherapy. Some methods treat a damaging effect of hemorrhagic stroke. Insome methods, the agent is administered before, during or afterendovascular repair of the hemorrhage. In some methods, the agent isadministered before, during or after treatment with other drugs for thetreatment of hemorrhages affecting the central nervous system.

The invention further provides an agent that inhibits binding of PSD95to an NMDAR2 subunit for use in treatment of an ischemic stroke thattransforms to a hemorrhagic stroke as a result of reperfusion, whereinthe agent inhibits damaging effects of the ischemic and hemorrhagicstroke.

The invention further provides methods of treating a subject populationpresenting sign(s) and/or symptom(s) of ischemia, comprisingadministering an agent, which agent inhibits binding of PSD-95 inhibitorto an NDMAR2 subunit or nNOS, to the subjects; wherein the subjects areanalyzed for unacceptable risk of side effects of reperfusion therapy,and subjects without unacceptable risk of side effects receivereperfusion therapy and subjects with unacceptable risk of side effectsdo not receive reperfusion therapy, optionally wherein the agent is apeptide that inhibits binding of PSD95 to an NMDAR2 subunit or nNOS, andthe peptide is linked to an internalization peptide or is lipidated toincrease across a cell membrane or the blood brain barrier.

In any of the above methods or agent, the agent can be a peptide havingan amino acid sequence consisting or comprising of X₁tSX₂V (SEQ IDNO:7), wherein t and S are alternative amino acids, X₁ is selected fromamong E, Q, and A, or an analogue thereof, X₂ is selected from among A,Q, D, N, (N-Methyl)-A, (N-methyl)-Q, (N-methyl)-D, and (N-methyl)-N oran analog thereof, and the peptide is linked at its N-terminal aminoacid to an internalization peptide. In any of the above methods andagents, the agent can have an amino acid sequence consisting orcomprising of YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6) or YGRKKRRQRRRKLSSIETDV(SEQ ID NO:37). In any of the above methods, the peptide or other agentcan be linked to an internalization peptide or lipidated therebyfacilitating passage of the peptide across a cell membrane or the bloodbrain barrier. Some peptides or other agents are myristoylated. Peptidesare preferably myristoylated at the N-terminus.

The invention further provides methods of screening a compound foractivity useful in treating or effecting prophylaxis of stroke orhemorrhage in or otherwise affecting the CNS, comprising administeringthe compound to humans undergoing an endovascular repair procedureaffecting the central nervous system, and determining whether thecompound reduces the number of infarcts observed by MRI compared with anegative control. In some methods, the MRI imaging includes DWI MRI. Insome methods, the MRI imaging includes FLAIR MRI. In some methods, theMRI imaging includes DWI and FLAIR MRI and infarcts resulting from theendovascular procedure are determined by identifying infarcts present onboth DWI and FLAIR MRI.

The invention further provides an isolated peptide having an amino acidsequence consisting or comprising of X₁tSX₂V (SEQ ID NO:7), wherein tand S are alternative amino acids, X₁ is selected from among E, Q, andA, X₂ is selected from among A, Q, D, N, (N-Methyl)-A, (N-methyl)-Q,(N-methyl)-D, and (N-methyl)-N, wherein the peptide is lipidated at theN-terminal amino acid. Some peptides have an amino acid sequencecomprising or consisting of KLSSIESDV or KLSSIETDV.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Sample DWI and FLAIR MRI scans of a single patient taken onenrollment (Pre-procedure) and on day 2 after the endovascular procedure(Post-procedure). Arrows indicate examples of DWI-positive embolicstrokes (yellow; upper panels) and their FLAIR-positive counterparts(red; lower panels). Rightmost panels (ROIs) indicate selection of theregions of interest used to count the numbers and calculate the volumesof the ischemic lesions.

FIG. 1B, C: Distribution of DWI MRI-detectable lesion numbers and lesionvolumes for all randomized patients who underwent a day 2-4 MRI (B;n=184), and for patients whose strokes were <10cc's (C; n=182). Due tothe extremely skewed distribution of the data, means and their standarddeviations are expected to be highly affected by outliers. Insets in Bprovide representative slices of DWI MRI scans from the two patients whoexperienced large strokes (>10cc's) as a result of proceduralcomplications. As the counts of lesions are independent of volume,counts are less sensitive to large strokes.

FIG. 2. Numbers of patients who were screened, randomly assigned to astudy group, and included in the per-protocol population. The modifiedintention-to-treat population was defined as all patients who wereenrolled and randomly assigned to a study group and who received studydrug (TAT-NR2B9C or Placebo). The per-protocol population was defined asall randomly assigned patients who received TAT-NR2B9C or placebo andwho were not excluded because of major protocol violations, whichincluded the inability to complete the post-endovascular procedure MRIscans (1 patient, who died before a scan could be done), or lack ofattendance at the 30 day end-of-study visit due to death or refusal toreturn for follow-up. However, of the 185 patients who were randomized,184 completed their day 2-4 post-procedure MRI scans, and these wereincluded in the analysis.

DEFINITIONS

A “chimeric peptide” means a peptide having two component peptides notnaturally associated with one another joined to one another as a fusionprotein or by chemical linkage.

A “fusion” protein or polypeptide refers to a composite polypeptide,i.e., a single contiguous amino acid sequence, made up of sequences fromtwo (or more) distinct, heterologous polypeptides which are not normallyfused together in a single polypeptide sequence.

The term “PDZ domain” refers to a modular protein domain of about 90amino acids, characterized by significant sequence identity (e.g., atleast 60%) to the brain synaptic protein PSD-95, the Drosophila septatejunction protein Discs-Large (DLG), and the epithelial tight junctionprotein ZO1 (ZO1). PDZ domains are also known as Discs-Large homologyrepeats (“DHRs”) and GLGF (SEQ ID NO:68) repeats. PDZ domains generallyappear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell85: 1067-76). Exemplary PDZ domain-containing proteins and PDZ domainsequences disclosed in U.S. application Ser. No. 10/714,537, which isherein incorporated by reference in its entirety.

The term “PL protein” or “PDZ Ligand protein” refers to a naturallyoccurring protein that forms a molecular complex with a PDZ-domain, orto a protein whose carboxy-terminus, when expressed separately from thefull length protein (e.g., as a peptide fragment of 3-25 residues, e.g.3, 4, 5, 8, 9, 10, 12, 14 or 16 residues), forms such a molecularcomplex. The molecular complex can be observed in vitro using the “Aassay” or “G assay” described, e.g., in U.S. application Ser. No.10/714,537, or in vivo.

The term “NMDA receptor,” or “NMDAR,” refers to a membrane associatedprotein that is known to interact with NMDA including the varioussubunit forms described below. Such receptors can be human or non-human(e.g., mouse, rat, rabbit, monkey).

A “PL motif” refers to the amino acid sequence of the C-terminus of a PLprotein (e.g., the C-terminal 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or25 contiguous residues) (“C-terminal PL sequence”) or to an internalsequence known to bind a PDZ domain (“internal PL sequence”).

A “PL peptide” is a peptide of comprising or consisting of, or otherwisebased on, a PL motif that specifically binds to a PDZ domain.

The terms “isolated” or “purified” means that the object species (e.g.,a peptide) has been purified from contaminants that are present in asample, such as a sample obtained from natural sources that contain theobject species. If an object species is isolated or purified it is thepredominant macromolecular (e.g., polypeptide) species present in asample (i.e., on a molar basis it is more abundant than any otherindividual species in the composition), and preferably the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, an isolated, purified orsubstantially pure composition comprises more than 80 to 90 percent ofall macromolecular species present in a composition. Most preferably,the object species is purified to essential homogeneity (i.e.,contaminant species cannot be detected in the composition byconventional detection methods), in which the composition consistsessentially of a single macromolecular species. The term isolated orpurified does not necessarily exclude the presence of other componentsintended to act in combination with an isolated species. For example, aninternalization peptide can be described as isolated notwithstandingthat it is linked to an active peptide or combined with apharmaceutically acceptable excipient.

A “peptidomimetic” refers to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics of apeptide consisting of natural amino acids. The peptidomimetic cancontain entirely synthetic, non-natural analogues of amino acids, or canbe a chimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The peptidomimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or inhibitory or binding activity.

Individual peptidomimetic residues can be joined by peptide bonds, otherchemical bonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,NjN-dicyclohexylcarbodiimide (DCC) or N₅N-diisopropylcarbodiimide (DIC).Linking groups that can be an alternative to the traditional amide bond(“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂- for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄.—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, A Peptide Backbone Modifications, Marcell Dekker, N.Y.).

Mimetics of aromatic amino acids can be generated by replacing by, e.g.,D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine;D- or L-I, -2,3-, or A-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluorophenylalanine; D- or L-p-biphenylphenylalanine; K- orL-p-methoxybiphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anonnatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R—N—C—N—R═) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide orl-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions.

Mimetics of basic amino acids can be generated by substitution with,e.g., (in addition to lysine and arginine) the amino acids ornithine,citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid,where alkyl is defined above. Nitrile derivative (e.g., containing theCN-moiety in place of COOH) can be substituted for asparagine orglutamine. Asparaginyl and glutaminyl residues can be deaminated to thecorresponding aspartyl or glutamyl residues.

Arginine residue mimetics can be generated by reacting arginyl with,e.g., one or more conventional reagents, including, e.g., phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably underalkaline conditions.

Tyrosine residue mimetics can be generated by reacting tyrosyl with,e.g., aromatic diazonium compounds or tetranitromethane.N-acetylimidizol and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole.

Lysine mimetics can be generated (and amino terminal residues can bealtered) by reacting lysinyl with, e.g., succinic or other carboxylicacid anhydrides. Lysine and other alpha-amino-containing residuemimetics can also be generated by reaction with imidoesters, such asmethyl picolinimidate, pyridoxal phosphate, pyridoxal,chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4,pentanedione, and transamidase-catalyzed reactions with glyoxylate.

Mimetics of methionine can be generated by reaction with, e.g.,methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid,thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline,3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residuemimetics can be generated by reacting histidyl with, e.g.,diethylprocarbonate or para-bromophenacyl bromide.

Other mimetics include, e.g., those generated by hydroxylation ofproline and lysine; phosphorylation of the hydroxyl groups of seryl orthreonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

The peptidomimetics of the invention can also include a structuralmimetic residue, particularly a residue that induces or mimics secondarystructures, such as a beta turn, beta sheet, alpha helix structures,gamma turns, and the like. For example, substitution of natural aminoacid residues with D-amino acids; N-alpha-methyl amino acids;C-alpha-methyl amino acids; or dehydroamino acids within a peptide caninduce or stabilize beta turns, gamma turns, beta sheets or alpha helixconformations. Beta turn mimetic structures have been described, e.g.,by Nagai (1985) Tet. Lett. 26:647-650; Feigl (1986) J. Amer. Chem. Soc.108:181-182; Kahn (1988) J. Amer. Chem. Soc. 110:1638-1639; Kemp (1988)Tet. Lett. 29:5057-5060; Kahn (1988) J. Molec. Recognition 1:75-79. Betasheet mimetic structures have been described, e.g., by Smith (1992) J.Amer. Chem. Soc. 114:10672-10674. For example, a type VI beta turninduced by a cis amide surrogate, 1,5-disubstituted tetrazol, isdescribed by Beusen (1995) Biopolymers 36:181-200. Incorporation ofachiral omega-amino acid residues to generate polymethylene units as asubstitution for amide bonds is described by Banerjee (1996) Biopolymers39:769-777′. Secondary structures of polypeptides can be analyzed by,e.g., high-field .sup. IH NMR or 2D NMR spectroscopy, see, e.g., Higgins(1997) J. Pept. Res. 50:421-435. See also, Hruby (1997) Biopolymers43:219-266, Balaji, et al., U.S. Pat. No. 5,612,895.

Peptidomimetics can contain any combination of nonnatural structuralcomponents, which are typically from three structural groups: a) residuelinkage groups other than the natural amide bond (“peptide bond”)linkages; b) non-natural residues in place of naturally occurring aminoacid residues; or c) residues which induce secondary structural mimicry,i.e., to induce or stabilize a secondary structure, e.g., a beta turn,gamma turn, beta sheet, alpha helix conformation, and the like. In apeptidomimetic of a chimeric peptide comprising an active peptide and aninternalization peptide, either the active moiety or the internalizationmoiety or both can be a peptidomimetic.

The term “specific binding” refers to binding between two molecules, forexample, a ligand and a receptor, characterized by the ability of amolecule (ligand) to associate with another specific molecule (receptor)even in the presence of many other diverse molecules, i.e., to showpreferential binding of one molecule for another in a heterogeneousmixture of molecules. Specific binding of a ligand to a receptor is alsoevidenced by reduced binding of a detectably labeled ligand to thereceptor in the presence of excess unlabeled ligand (i.e., a bindingcompetition assay).

Excitotoxicity is the pathological process by which neurons are damagedand killed by the overactivation of receptors for the excitatoryneurotransmitter glutamate, such as the NMDA receptors, e.g., NMDAR2B.

The term “subject” includes humans and veterinary animals, such asmammals, as well as laboratory animal models, such as mice or rats usedin preclinical studies.

The term “agent” includes any compound including compounds with orwithout pharmaceutical activity, natural compounds, synthetic compounds,small molecules, peptides and peptidomimetics.

The term “pharmacologic agent” means an agent having a pharmacologicalactivity. Pharmacological agents include compounds that are known drugs,compounds for which pharmacological activity has been identified butwhich are undergoing further therapeutic evaluation in animal models orclinical trials. A chimeric agent comprises a pharmacologic agent linkedto an internalization peptide. An agent can be described as havingpharmacological activity if it exhibits an activity in a screeningsystem that indicates that the active agent is or may be useful in theprophylaxis or treatment of a disease. The screening system can be invitro, cellular, animal or human. Agents can be described as havingpharmacological activity notwithstanding that further testing may berequired to establish actual prophylactic or therapeutic utility intreatment of a disease.

A tat peptide means a peptide comprising or consisting of GRKKRRQRRR(SEQ ID NO:1), in which no more than 5 residues are deleted, substitutedor inserted within the sequence, which retains the capacity tofacilitate uptake of a linked peptide or other agent into cells.Preferably any amino acid changes are conservative substitutions.Preferably, any substitutions, deletions or internal insertions in theaggregate leave the peptide with a net cationic charge, preferablysimilar to that of the above sequence. Such can be accomplished by notsubstituting or deleting the R and K residues. The amino acids of a tatpeptide can be derivatized with biotin or similar molecule to reduce aninflammatory response.

Co-administration of a pharmacological agents means that the agents areadministered sufficiently close in time for detectable amounts of theagents to present in the plasma simultaneously and/or the agents exert atreatment effect on the same episode of disease or the agents actco-operatively or synergistically in treating the same episode ofdisease. For example, an anti-inflammatory agent acts cooperatively withan agent including a tat peptide when the two agents are administeredsufficiently proximately in time that the anti-inflammatory agent caninhibit an inflammatory response inducible by the internationalizationpeptide.

Statistically significant refers to a p-value that is <0.05, preferably<0.01 and most preferably <0.001.

An episode of a disease means a period when signs and/or symptoms of thedisease are present interspersed by flanked by longer periods in whichthe signs and/or symptoms or absent or present to a lesser extent.

The CNS is used in accordance with convention to mean the brain and/orspinal cord. Ischemia or hemorrhage can affect the CNS if it occurs inthe CNS, immediately proximal to the CNS, such as a subarachnoidhemorrhage or more distally within a blood vessel supplying the CNS.Ischemia or hemorrhage affects the CNS, if it of a type when leftuntreated, it causes detectable pathology in the CNS or a neurocognitivedeficit.

In conditions such as SAH resulting from rupture, rupture is consideredto occur on day 1. Thus, for example if rupture occurs on a Monday andtreatment occurs within four days of rupture, treatment occurs by theend of Friday. Treatment occurring with days 5-12 post-rupture occurswithin the period of Saturday to the following Friday.

The invention also provides methods of treating a damaging effect ofsubarachnoid hemorrhage in a population of subjects, comprising:administering an agent that inhibits binding of PSD-95 to an NMDAR2subunit to subjects having a subarachnoid hemorrhage, wherein thedamaging effect is reduced in the administered population compared tocontrol subjects not receiving the agent. The damaging effect that isreduced can be neuronal cell death or a cognitive deficit.

DETAILED DESCRIPTION I. General

The present application provides data from a clinical trial of a PSD-95inhibitor in subjects undergoing endovascular repair of an aneurysm inor otherwise affecting the CNS. The subjects were stratified by whetherthe aneurysm ruptured before performing the endovascular surgery.Rupture is associated with higher mortality or increased debilitation ifa subject survives. Outcome was assessed by number and volume ofinfarctions and neurocognitive measures. The trial provided evidence ofsignificant benefit in subjects harboring an intracranial aneurysm inneed of endovascular repair whether or not the aneurysm ruptured beforeendovascular was surgery performed, and showed minimal side effects.Surprisingly, however, the subjects benefitting most from treatment asjudged both by pathology and neurocognitive outcome were those in whichthe aneurysm had ruptured prior to the endovascular procedure causing asubarachnoid hemorrhage. These data constitute evidence that a PSD-95inhibitor is beneficial not only in ischemic and hemorrhagic stroke butin forms of hemorrhage in or affecting the CNS whether or not resultingfrom stroke including cerebral hemorrhage, intracerebral hemorrhage,intracranial hemorrhage (ICH), neurotrauma, traumatic brain injury andsubdural and epidural hemorrhages, and particularly, subarachnoidhemorrhage (SAH). That treatment with a PSD-95 inhibitor is effective inboth ischemic and hemorrhagic injury to the CNS, and has minimal sideeffects, means that such an inhibitor can be given to any subjectpresenting with signs of stroke or hemorrhage affecting the CNS withoutdelaying treatment by first performing a detailed diagnostic work up,typically a brain scan, to distinguish between ischemia and hemorrhage.The trial also showed that a PSD-95 inhibitor was effective in reducingpain sometimes associated with endovascular surgery along the path ofthe groin puncture necessary to insert the endovascular catheters intothe arterial system and traverse to its site of action, in this case, apath from the groin area to the brain area (e.g., leg, groin, abdominalarea, chest, neck, and head). Although PSD-95 inhibitors have previouslybeen reported effective in treating some forms of pain, the result thatthe same administration of a PSD-95 inhibitor has a dual action ininhibiting damage resulting from endovascular repair of an aneurysm andthe type of pain caused by the endovascular surgery itself was notknown.

II. Agents Inhibiting PSD-95

Such agents inhibit interactions between PSD-95 and one or more NMDARs,e.g., by specifically binding to PSD-95. Preferably, inhibition is of anNMDAR2 (e.g., 2A, 2B, 2C or 2D). Reference to such agents can refer tothe agents alone or more typically in the case of peptide agents, theagents linked to an internalization peptide as a chimeric peptide. Suchagents are useful for reducing one or more damaging effects of strokeand other neurological conditions mediated at least in part by NMDARexcitotoxicity. Such agents include peptides having an amino acidsequence including or based on the PL motif of a NMDA Receptor or PDZdomain of PSD-95. Such peptides can also inhibit interactions betweenPSD-95 and nNOS and other glutamate receptors (e.g., kainite receptorsor AMPA receptors), such as KV1-4 and GluR6. Preferred peptides inhibitinteraction between PDZ domains 1 and 2 of postsynaptic density-95protein (PSD-95)(human amino acid sequence provided by Stathakism,Genomics 44(1):71-82 (1997)) and the C-terminal PL sequence of one ormore NMDA Receptor 2 subunits including the NR2B subunit of the neuronalN-methyl-D-aspartate receptor (Mandich et al., Genomics 22, 216-8(1994)). NMDAR2B has GenBank ID 4099612, a C-terminal 20 amino acidsFNGSSNGHVYEKLSSIESDV (SEQ ID NO:11) and a PL motif ESDV (SEQ ID NO:12).Preferred peptides inhibit the human forms of PSD-95 and human NMDARreceptors. However, inhibition can also be shown from species variantsof the proteins. A list of NMDA and glutamate receptors that can be usedappears below.

TABLE 1 NMDA Receptors With PL Sequences C-terminal C-terminal NameGI or Acc# 20mer sequence 4mer sequence PL? NMDAR1 307302HPTDITGPLNLSDPSVSTVV STVV X (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1-1292282 HPTDITGPLNLSDPSVSTVV STVV X (SEQ ID NO: 13) (SEQ ID NO: 27)NMDAR1-4 472845 HPTDITGPLNLSDPSVSTVV STVV X (SEQ ID NO: 13)(SEQ ID NO: 27) NMDAR1-3b 2343286 HPTDITGPLNLSDPSVSTVV STVV X(SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1-4b 2343288 HPTDITGPLNLSDPSVSTVVSTVV X (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1-2 11038634RRAIEREEGQLQLCSRHRES HRES (SEQ ID NO: 14) (SEQ ID NO: 28) NMDAR1-311038636 RRAIEREEGQLQLCSRHRES HRES (SEQ ID NO: 14) (SEQ ID NO: 28)NMDAR2C 6006004 TQGFPGPCTWRRISSLESEV ESEV X (SEQ ID NO: 15)(SEQ ID NO: 29) NMDAR3 560546 FNGSSNGHVYEKLSSIESDV ESDV X(SEQ ID NO: 11) (SEQ ID NO: 12) NMDAR3A 17530176 AVSRKTELEEYQRTSRTCESICES (SEQ ID NO: 16) (SEQ ID NO: 30) NMDAR2B 4099612FNGSSNGHVYEKLSSIESDV ESDV X (SEQ ID NO: 11) (SEQ ID NO: 12) NMDAR2A558748 LNSCSNRRVYKKMPSIESDV ESDV X (SEQ ID NO: 17) (SEQ ID NO: 12)NMDAR2D 4504130 GGDLGTRRGSAHFSSLESEV ESEV X (SEQ ID NO: 18)(SEQ ID NO: 29) Glutamate AF009014 QPIPTLGLNLGNDPDRGTSI GISI (SEQ ID Xreceptor (SEQ ID NO: 19) NO: 31) delta 2 Glutamate 128953MOSIPCMSHSSGMPLGATGL ATGL (SEQ ID X receptor 1 (SEQ ID NO: 20) NO: 32)Glutamate L20814 QNFATYKEGYNVYGIESVKI SVKI (SEQ ID X receptor 2(SEQ ID NO: 21) NO: 33) Glutamate AF167332 QNYATYREGYNVYGTESVKISVKI (SEQ ID X receptor 3 (SEQ ID NO: 22) NO: 33) Glutamate U16129HTGTAIRQSSGLAVIASDLP SDLP (SEQ ID receptor 4 (SEQ ID NO: 23) NO: 34)Glutamate U16125 SFTSILTCHQRRTQRKETVA ETVA (SEQ ID X receptor 5(SEQ ID NO: 24) NO: 35) Glutamate U16126 EVINMHTFNDRRLPGKETMAETMA (SEQ ID X receptor 6 (SEQ ID NO: 25) NO: 36)

Some peptides inhibit interactions between PSD-95 and multiple NMDARsubunits. In such instances, use of the peptide does not necessarilyrequire an understanding of the respective contributions of thedifferent NMDARs to excitatory neurotransmission. Other peptides arespecific for a single NMDAR.

Peptides can include or be based on a PL motif from the C-terminus ofany of the above subunits and have an amino acid sequence comprising[S/T]-X-[V/L]. This sequence preferably occurs at the C-terminus of thepeptides of the invention. Preferred peptides have an amino acidsequence comprising [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:38) attheir C-terminus. Exemplary peptides comprise: ESDV (SEQ ID NO:12), ESEV(SEQ ID NO:29), ETDV (SEQ ID NO:39), ETEV (SEQ ID NO:40), DTDV (SEQ IDNO:41), and DTEV (SEQ ID NO:42) as the C-terminal amino acids. Twoparticularly preferred peptides have an amino acid sequence comprisingor consisting of KLSSIESDV (SEQ ID NO:5), and KLSSIETDV (SEQ ID NO:43).Such peptides usually have 3-25 amino acids (without an internalizationpeptide), peptide lengths of 5-10 amino acids, and particularly 9 aminoacids (also without an internalization peptide) are preferred. In somesuch peptides, all amino acids are from the C-terminus of an NMDAreceptor (not including amino acids from an internalization peptide).The invention also includes peptidomimetics of these and other peptidesdisclosed herein.

Other peptides that inhibit interactions between PDS95 and NDMARsinclude peptides from PDZ domain 1 and/or 2 of PSD-95 or a subfragmentof any of these that inhibits interactions between PSD-95 and an NMDAreceptor, such as NR2B. Such active peptides comprise at least 50, 60,70, 80 or 90 amino acids from PDZ domain 1 and/or PDZ domain 2 ofPSD-95, which occur within approximately amino acids 65-248 of PSD-95provided by Stathakism, Genomics 44(1):71-82 (1997) (human sequence) orNP_031890.1, GI:6681195 (mouse sequence) or corresponding regions ofother species variants.

Peptides and peptidomimetics of the invention can contain modified aminoacid residues for example, residues that are N-alkylated. N-terminalalkyl modifications can include e.g., N-Methyl, N-Ethyl, N-Propyl,N-Butyl, N-Cyclohexylmethyl, N-Cyclyhexylethyl, N-Benzyl, N-Phenylethyl,N-phenylpropyl, N-(3, 4-Dichlorophenyl)propyl,N-(3,4-Difluorophenyl)propyl, and N-(Naphthalene-2-yl)ethyl).

Bach, J. Med. Chem. 51, 6450-6459 (2008) and WO 2010/004003 hasdescribed a series of analogs of NR2B9c. PDZ-binding activity isexhibited by peptides having only three C-terminal amino acids (SDV).Bach also reports analogs having an amino acid sequence comprising orconsisting of X₁tSX₂V (SEQ ID NO:7), wherein t and S are alternativeamino acids, X₁ is selected from among E, Q, and A, or an analoguethereof, X2 is selected from among A, Q, D, N, (N-Methyl)-A,(N-methyl)-Q, (N-methyl)-D, and (N-methyl)-N or an analogue thereof.Optionally the peptide is N-alkylated in position P3 position (thirdamino acid from C-terminus, i.e. position occupied by tS). The peptidecan be N-alkylated with a cyclohexane or aromatic substituent, andfurther comprises a spacer group between the substituent and theterminal amino group of the peptide or peptide analogue, wherein thespacer is an alkyl group, preferably selected from among methylene,ethylene, propylene and butylene. The aromatic substituent can be anaphthalen-2-yl moiety or an aromatic ring substituted with one or twohalogen and/or alkyl group.

Other modifications can also be incorporated without adversely affectingthe activity and these include substitution of one or more of the aminoacids in the natural L-isomeric form with amino acids in the D-isomericform. Thus, any amino acid naturally occurring in the L-configuration(which can also be referred to as the R or S, depending upon thestructure of the chemical entity) can be replaced with the amino acid ofthe same chemical structural type or a peptidomimetic, but of theopposite chirality, generally referred to as the D-amino acid, but whichcan additionally be referred to as the R- or S-form. Thus, apeptidomimetic may include 1, 2, 3, 4, 5, at least 50%, or all D-aminoacid resides. A peptidomimetic containing some or all D residues issometimes referred to an “inverso” peptide.

Peptidomimetics also include retro peptides. A retro peptide has areverse amino acid sequence. Peptidomimetics also include retro inversopeptides in which the order of amino acids is reversed from so theoriginally C-terminal amino acid appears at the N-terminus and D-aminoacids are used in place of L-amino acids. WO 2008/014917 describes aretro-inverso analog of Tat-NR2B9c having the amino acid sequencevdseisslk-rrrqrrkkrgyin (SEQ ID NO:8) (lower case letters indicating Damino acids), and reports it to be effective inhibiting cerebralischemia. Another effect peptide described herein is Rv-Tat-NR2B9c(RRRQRRKKRGYKLSSIESDV; SEQ ID NO:9).

A linker, e.g., a polyethylene glycol linker, can be used to dimerizethe active moiety of the peptide or the peptidomimetic to enhance itsaffinity and selectivity towards proteins containing tandem PDZ domains.See e.g., Bach et al., (2009) Angew. Chem. Int. Ed. 48:9685-9689 and WO2010/004003. A PL motif-containing peptide is preferably dimerized viajoining the N-termini of two such molecules, leaving the C-termini free.Bach further reports that a pentamer peptide IESDV (SEQ ID NO:10) fromthe C-terminus of NMDAR2B was effective in inhibiting binding of NMDAR2Bto PSD-95. IETDV (SEQ ID NO:73) can also be used instead of IESDV as canpeptides comprising IESDV or IESTDV. Optionally, about 2-10 copies of aPEG can be joined in tandem as a linker. Optionally, the linker can alsobe attached to an internalization peptide or lipidated to enhancecellular uptake. Examples of illustrative dimeric inhibitors are shownbelow (see Bach et al., PNAS 109 (2012) 3317-3322). Any of the PSD-95inhibitors disclosed herein can be used instead of IETDV, and anyinternalization peptide or lipidating moiety can be used instead of tatin the configurations shown below. Other linkers to that shown can alsobe used.

IETAV is assigned SEQ ID NO:74, YGRKKRRQRRR SEQ ID NO:2, and rrrqrrkkr,SEQ ID NO:75, lower case letters indicated D-amino acids.

Appropriate pharmacological activity of peptides, peptidomimetics orother agent can be confirmed if desired, using previously described ratmodels of stroke before testing in the primate and clinical trialsdescribed in the present application. Peptides, peptidomimetics or otheragents can also be screened for capacity to inhibit interactions betweenPSD-95 and NMDAR2B using assays described in e.g., US 20050059597, whichis incorporated by reference. Useful peptides, peptidomimetics or otheragents typically have IC50 values of less than 50 μM, 25 μM, 10 μM, 0.1μM or 0.01 μM in such an assay. Preferred peptides typically have anIC50 value of between 0.001-1 μM, and more preferably 0.05-0.5 or 0.05to 0.1 μM. When a peptide, peptidomimetic or other agent ischaracterized as inhibiting binding of one interaction, e.g., PSD-95interaction to NMDAR2B, such description does not exclude that thepeptide or agent also inhibits another interaction, for example,inhibition of PSD-95 binding to nNOS.

Peptides such as those just described can optionally be derivatized(e.g., acetylated, phosphorylated and/or glycosylated) to improve thebinding affinity of the inhibitor, to improve the ability of theinhibitor to be transported across a cell membrane or to improvestability. As a specific example, for inhibitors in which the thirdresidue from the C-terminus is S or T, this residue can bephosphorylated before use of the peptide.

Pharmacological agents also include small molecules that inhibitinteractions between PSD-95 and NMDAR2B, and/or other interactionsdescribed above. Suitable small-molecule inhibitors are described inWO/2009/006611. An exemplary class of suitable compounds are of theformula:

wherein R¹ is a member selected from the group consisting of cyclohexylsubstituted with 0-4 R⁷, phenyl substituted with 0-4 R⁷,—(CH₂)_(u)-(CHR⁸R⁹), a branched C₁₋₆ alkyl (isopropyl, isobutyl,1-isopropyl-2-methyl-butyl, 1 ethyl-propyl), and—NH—C(O)—(CR¹⁰R¹¹)_(v)H;

each R⁷ is independently a member selected from the group consisting ofC₁₋₆ alkyl, C₁₋₆ alkoxy, —C(O)R¹², OH, COOH, —NO, N-substituted indolineand a cell membrane translocation peptide;

each R⁸ and R⁹ is independently selected from the group consisting of H,OH, cyclohexane, cyclopentane, phenyl, substituted phenyl andcyclopentadiene;

each R¹⁰ and R¹¹ is independently selected from the group consisting ofH, cyclohexane, phenyl and a cell membrane translocation peptide;

R¹² is a member selected from the group consisting of C₁₋₆ alkyl andaryl; and each of u and v are independently from 0 to 20;

wherein one of R², R³, R⁴, R⁵ and R⁶ is —COOH, and wherein the remainderof R², R³, R⁴, R⁵ and R⁶ are each independently selected from the groupconsisting of F, H, OCH₃ and CH₃.

One such compound is 0620-0057, the structure of which is:

A pharmacological agent can be linked to an internalization peptide tofacilitate uptake into cells and/or across the blood brain barrier. Anyof the above pharmacological agents can be linked to any of theinternalization peptides described below. Internalization peptides are awell-known class of relatively short peptides that allow many cellularor viral proteins to traverse membranes. Internalization peptides, alsoknown as cell membrane transduction peptides or cell penetratingpeptides can have e.g., 5-30 amino acids. Such peptides typically have acationic charge from an above normal representation (relative toproteins in general) of arginine and/or lysine residues that is believedto facilitate their passage across membranes. Some such peptides have atleast 5, 6, 7 or 8 arginine and/or lysine residues. Examples include theantennapedia protein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (andvariants thereof), the tat protein of human immunodeficiency virus, theprotein VP22, the product of the UL49 gene of herpes simplex virus type1, Penetratin, SynB1 and 3, Transportan, Amphipathic, gp41NLS, polyArg,and several plant and bacterial protein toxins, such as ricin, abrin,modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat labiletoxins, and Pseudomonas aeruginosa exotoxin A (ETA). Other examples aredescribed in the following references (Temsamani, Drug Discovery Today,9(23):1012-1019, 2004; De Coupade, Biochem J., 390:407-418, 2005; SaalikBioconjugate Chem. 15: 1246-1253, 2004; Zhao, Medicinal Research Reviews24(1):1-12, 2004; Deshayes, Cellular and Molecular Life Sciences62:1839-49, 2005, Gao, ACS Chem. Biol. 2011, 6, 484-491, SG3(RLSGMNEVLSFRWL) (all incorporated by reference).

A preferred internalization peptide is tat from the HIV virus. A tatpeptide reported in previous work comprises or consists of the standardamino acid sequence YGRKKRRQRRR (SEQ ID NO:2) found in HIV Tat protein.Thus two preferred agents incorporating this tat peptide are thepeptides comprising or consisting of the amino acid sequenceYGRKKRRQRRRKLSSIESDV, SEQ ID NO:6 (also known as Tat-NR2B9c orTAT-NR2B9C) or YGRKKRRQRRRKLSSIETDV (SEQ ID NO:37). If additionalresidues flanking such a tat motif are present (beside thepharmacological agent) the residues can be for example natural aminoacids flanking this segment from a tat protein, spacer or linker aminoacids of a kind typically used to join two peptide domains, e.g., gly(ser)₄ (SEQ ID NO:44), TGEKP (SEQ ID NO:45), GGRRGGGS (SEQ ID NO:46), orLRQRDGERP (SEQ ID NO:47) (see, e.g., Tang et al. (1996), J. Biol. Chem.271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11, 405-410)), orcan be any other amino acids that do not significantly reduce capacityto confer uptake of the variant without the flanking residues.Preferably, the number of flanking amino acids other than an activepeptide does not exceed ten on either side of YGRKKRRQRRR (SEQ ID NO:2).One suitable tat peptide comprising additional amino acid residuesflanking the C-terminus of YGRKKRRQRRR (SEQ ID NO:2) is YGRKKRRQRRRPQ(SEQ ID NO:48). However, preferably, no flanking amino acids arepresent. Other tat peptides that can be used include GRKKRRQRRRPQ (SEQID NO:4) and GRKKRRQRRRP (SEQ ID NO:26).

Variants of the above tat peptide having reduced capacity to bind toN-type calcium channels are described by WO/2008/109010. Such variantscan comprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ IDNO:49), in which X is an amino acid other than Y or nothing (in whichcase G is a free N-terminal residue). A preferred tat peptide has theN-terminal Y residue substituted with F. Thus, a tat peptide comprisingor consisting of FGRKKRRQRRR (SEQ ID NO:3) is preferred. Anotherpreferred variant tat peptide consists of GRKKRRQRRR (SEQ ID NO:1).Another preferred tat peptide comprises or consists of RRRQRRKKRG orRRRQRRKKRGY (amino acids 1-10 or 1-11 of SEQ ID NO:9). Other tat derivedpeptides that facilitate uptake of a pharmacological agent withoutinhibiting N-type calcium channels include those presented in Table 2below.

TABLE 2 X-FGRKKRRQRRR (F-Tat) (SEQ ID NO: 69)X-GKKKKKQKKK (SEQ ID NO: 50) X-RKKRRQRRR (SEQ ID NO: 51)X-GAKKRRQRRR (SEQ ID NO: 52) X-AKKRRQRRR (SEQ ID NO: 53)X-GRKARRQRRR (SEQ ID NO: 54) X-RKARRQRRR (SEQ ID NO: 55)X-GRKKARQRRR (SEQ ID NO: 56) X-RKKARQRRR (SEQ ID NO: 57)X-GRKKRRQARR (SEQ ID NO: 58) X-RKKRRQARR (SEQ ID NO: 59)X-GRKKRRQRAR (SEQ ID NO: 60) X-RKKRRQRAR (SEQ ID NO: 61)X-RRPRRPRRPRR (SEQ ID NO: 62) X-RRARRARRARR (SEQ ID NO: 63)X-RRRARRRARR (SEQ ID NO: 64) X-RRRPRRRPRR (SEQ ID NO: 65)X-RRPRRPRR (SEQ ID NO: 66) X-RRARRARR (SEQ ID NO: 67)

X can represent a free amino terminus, one or more amino acids, or aconjugated moiety. Internalization peptides can be used in inverso orretro or inverso retro form with or without the linked peptide orpeptidomimetic being in such form. For example, a preferred chimericpeptide has an amino acid sequence comprising or consisting ofRRRQRRKKRGY-KLSSIESDV (SEQ ID NO:9) or having an amino acid sequencecomprising or consisting of RRRQRRKKRGY-KLSSIETDV (SEQ ID NO:37) .

Internalization peptides can be attached to pharmacological agents byconventional methods. For example, the agents can be joined tointernalization peptides by chemical linkage, for instance via acoupling or conjugating agent. Numerous such agents are commerciallyavailable and are reviewed by S. S. Wong, Chemistry of ProteinConjugation and Cross-Linking, CRC Press (1991). Some examples ofcross-linking reagents include J-succinimidyl3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-phenylene) bismaleimide;N,N′-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11carbon methylene bridges (which relatively specific for sulfhydrylgroups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversiblelinkages with amino and tyrosine groups). Other cross-linking reagentsinclude p,p′-difluoro-m, m′-dinitrodiphenylsulfone (which formsirreversible cross-linkages with amino and phenolic groups); dimethyladipimidate (which is specific for amino groups);phenol-1,4-disulfonylchloride (which reacts principally with aminogroups); hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate (which reacts principally with amino groups);glutaraldehyde (which reacts with several different side chains) anddisdiazobenzidine (which reacts primarily with tyrosine and histidine).

For pharmacological agents that are peptides attachment to aninternalization peptide can be achieved by generating a fusion proteincomprising the peptide sequence fused, preferably at its N-terminus, toan internalization peptide.

Instead of or as well as linking a peptide (or other agent) inhibitingPSD-95 to an internalization peptide, such a peptide can be linked to alipid (lipidation) to increase hydrophobicity of the conjugate relativeto the peptide alone and thereby facilitate passage of the linkedpeptide across cell membranes and/or across the brain barrier.Lipidation is preferably performed on the N-terminal amino acid but canalso be performed on internal amino acids, provided the ability of thepeptide to inhibit interaction between PSD-95 and NMDAR 2B is notreduced by more than 50%. Preferably, lipidation is performed on anamino acid other than one of the four most C-terminal amino acids.Lipids are organic molecules more soluble in ether than water andinclude fatty acids, glycerides and sterols. Suitable forms oflipidation include myristoylation, palmitoylation or attachment of otherfatty acids preferably with a chain length of 10-20 carbons, such aslauric acid and stearic acid, as well as geranylation,geranylgeranylation, and isoprenylation. Lipidations of a type occurringin posttranslational modification of natural proteins are preferred.Lipidation with a fatty acid via formation of an amide bond to thealpha-amino group of the N-terminal amino acid of the peptide is alsopreferred. Lipidation can be by peptide synthesis including aprelipidated amino acid, be performed enzymatically in vitro or byrecombinant expression, by chemical crosslinking or chemicalderivatization of the peptide. Amino acids modified by myristoylationand other lipid modifications are commercially available.

Lipidation preferably facilitates passage of a linked peptide (e.g.,KLSSIETDV (SEQ ID NO:5), or KLSSIETDV (SEQ ID NO:43)) across a cellmembrane and/or the blood brain barrier without causing a transientreduction of blood pressure as has been found when a standard tatpeptide is administered at high dosage (e.g., at or greater than 3mg/kg), or at least with smaller reduction that than the same peptidelinked to a standard tat peptide.

Pharmacologic peptides, optionally fused to tat peptides, can besynthesized by solid phase synthesis or recombinant methods.Peptidomimetics can be synthesized using a variety of procedures andmethodologies described in the scientific and patent literature, e.g.,Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley &Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.

III. Agents and Methods for Reperfusion

Treatment of ischemic strokes with a PSD-95 inhibitor can be combinedwith reperfusion therapy. Such reperfusion can be achieved using theintravenous or intra-arterial administration of thrombolytic agents suchas tPA, streptokinase or urokinase, by using mechanical means to re-openblocked arteries, or by other means to enhance the collateralcirculation to an ischemic brain area. By administering the PSD-95inhibitor, there is more time available time to perform a brain scan todetermine presence of ischemic stroke, and then administer tPA oradminister another reperfusion therapy if appropriate. Thus, moresubjects with ischemic stroke can benefit from tPA treatment or fromanother therapy that enhances brain reperfusion and at the same timebenefit from treatment with a PSD-95 inhibitor.

Plaques, blood clots or any other particulate matter (collectively knownas emboli) causing ischemia can be dissolved removed or bypassed by bothpharmacological and physical means. The dissolving, removal of emboli orother obstructions to blood flow and consequent restoration of bloodflow is referred to as reperfusion. One class of agents acts bythrombolysis. These agents work by stimulating fibrinolysis by plasminthrough infusion of tissue plasminogen activator (tPA). Plasmin clearscross-linked fibrin mesh (the backbone of a clot), making the clotsoluble and subject to further proteolysis by other enzymes, andrestores blood flow in occluded blood vessels. Examples of thrombolyticagents include tissue plasminogen activator t-PA, alteplase (Activase®), reteplase (Retavase®), tenecteplase (TNKase®), anistreplase(Eminase®), streptokinase (Kabikinase®, Streptase®), and urokinase)(Abbokinase®).

Another class of drugs that can be used for reperfusion is vasodilators.These drugs act by relaxing and opening up blood vessels thus allowingblood to flow around an obstruction. Some examples of types ofvasodilators alpha-adrenoceptor antagonists (alpha-blockers),Angiotensin receptor blockers (ARBs), Beta₂-adrenoceptor agonists(β₂-agonists), calcium-channel blockers (CCBs), centrally actingsympatholytics, direct acting vasodilators, endothelin receptorantagonists, ganglionic blockers, nitrodilators, phosphodiesteraseinhibitors, potassium-channel openers, and renin inhibitors

Mechanical methods of reperfusion include angioplasty, catheterization,and artery bypass graft surgery, stenting, embolectomy, orendarterectomy. Such procedures restore plaque flow by mechanicalremoval of a plaque, holding a blood vessel open, so blood can flowaround a plaque or bypassing a plaque. Other methods of enhancingreperfusion include adjunctive devices such as intra-aortic balloons(CoAxiaNeuroFlo™) that divert the cardiac output of blood to thecerebral circulation, thereby increasing collateral perfusion to theischemic area (see clinicaltrials.gov/ct2/show/NCT00119717).

IV. Stroke

A stroke is a condition resulting from impaired blood flow in the CNSregardless of cause. Potential causes include embolism, hemorrhage andthrombosis. Some neuronal cells die immediately as a result of impairedblood flow. These cells release their component molecules includingglutamate, which in turn activates NMDA receptors, which raiseintracellular calcium levels, and intracellular enzyme levels leading tofurther neuronal cell death (the excitotoxicity cascade). The death ofCNS tissue is referred to as infarction. Infarction Volume (i.e., thevolume of dead neuronal cells resulting from stroke in the brain) can beused as an indicator of the extent of pathological damage resulting fromstroke. In some instances, strokes may arise due to multiple emboli, ordue to a generalized arteriopathy. In the former, the emboli may arisefrom the heart, such as in the case of endocarditis, atrialfibrillation, or cardiac valvular disease. In the latter, thearteriopathy may comprise an arteritis (an infectious or autoimmuneinflammation of the arteries). In such instances, multiple strokes mayarise in the brain, sometimes due to many small emboli. In suchinstances, another means to measure the extent of pathological damageresulting from stroke is to count the number of ischemic lesions. Thisapplies especially in the case of procedurally-induced strokes, such asstrokes incurred after endovascular repair of brain aneurysm. In thisinstance, the intravascular manipulation may liberate multiple embolithat can produce many strokes in the brain. The symptomatic effectdepends both on the volume of an infarction, the number of infarctionsand where in the brain it/they is/are located. Disability index can beused as a measure of symptomatic damage, such as the Rankin StrokeOutcome Scale (Rankin, Scott Med J;2:200-15 (1957)) and the BarthelIndex. The Rankin Scale is based on assessing directly the globalconditions of a subject as follows.

Table 3

-   0 No symptoms at all-   1 No significant disability despite symptoms; able to carry out all    usual duties and activities.-   2 Slight disability; unable to carry out all previous activities but    able to look after own affairs without assistance.-   3 Moderate disability requiring some help, but able to walk without    assistance-   4 Moderate to severe disability; unable to walk without assistance    and unable to attend to own bodily needs without assistance.-   5 Severe disability; bedridden, incontinent, and requiring constant    nursing care and attention.

The Barthel Index is based on a series of questions about the subject'sability to carry out 10 basic activities of daily living resulting in ascore between 0 and 100, a lower score indicating more disability(Mahoney et al., Maryland State Medical Journal 14:56-61 (1965)).

Alternatively stroke severity/outcomes can be measured using the NIHstroke scale, available at world wide webninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf.

The scale is based on the ability of a subject to carry out 11 groups offunctions that include assessments of the subject's level ofconsciousness, motor, sensory and language functions.

An ischemic stroke refers more specifically to a type of stroke thatcaused by blockage of blood flow to the brain. The underlying conditionfor this type of blockage is most commonly the development of fattydeposits lining the vessel walls. This condition is calledatherosclerosis. These fatty deposits can cause two types ofobstruction. Cerebral thrombosis refers to a thrombus (blood clot) thatdevelops at the clogged part of the vessel “Cerebral embolism” refersgenerally to a blood clot that forms at another location in thecirculatory system, usually the heart and large arteries of the upperchest and neck. A portion of the blood clot then breaks loose, entersthe bloodstream and travels through the brain's blood vessels until itreaches vessels too small to let it pass. A second important cause ofembolism is an irregular heartbeat, known as arterial fibrillation. Itcreates conditions in which clots can form in the heart, dislodge andtravel to the brain. Additional potential causes of ischemic stroke arehemorrhage, thrombosis, dissection of an artery or vein, a cardiacarrest, shock of any cause including hemorrhage, and iatrogenic causessuch as direct surgical injury to brain blood vessels or vessels leadingto the brain or cardiac surgery. Ischemic stroke accounts for about 83percent of all cases of stroke. Another cause of blood clots is theintravascular stasis of blood due to vascular lesions such as brainaneurysms, due to the intravascular introduction of endovascular tools,or due to thrombosis due to the intravascular introduction ofendovascular tools.

Transient ischemic attacks (TIAs) are minor or warning strokes. In aTIA, conditions indicative of an ischemic stroke are present and thetypical stroke warning signs develop. However, the obstruction (bloodclot) occurs for a short time and tends to resolve itself through normalmechanisms. Subjects undergoing heart surgery are at particular risk oftransient cerebral ischemic attack.

Hemorrhagic stroke accounts for about 17 percent of stroke cases. Itresults from a weakened vessel that ruptures and bleeds into thesurrounding brain. The blood accumulates and compresses the surroundingbrain tissue. The two general types of hemorrhagic strokes areintracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic strokeresult from rupture of a weakened blood vessel ruptures. Potentialcauses of rupture from a weakened blood vessel include a hypertensivehemorrhage, in which high blood pressure causes a rupture of a bloodvessel, or another underlying cause of weakened blood vessels such as aruptured brain vascular malformation including a brain aneurysm,arteriovenous malformation (AVM) or cavernous malformation. Hemorrhagicstrokes can also arise from a hemorrhagic transformation of an ischemicstroke which weakens the blood vessels in the infarct, or a hemorrhagefrom primary or metastatic tumors in the CNS which contain abnormallyweak blood vessels. Ischemic stroke can also transform in to hemorrhagicstroke as a result of reperfusion. Hemorrhagic stroke can also arisefrom iatrogenic causes such as direct surgical injury to a brain bloodvessel. An aneurysm is a ballooning of a weakened region of a bloodvessel. If left untreated, the aneurysm continues to weaken until itruptures and bleeds into the brain. An arteriovenous malformation (AVM)is a cluster of abnormally formed blood vessels. A cavernousmalformation is a venous abnormality that can cause a hemorrhage fromweakened venous structures. Any one of these vessels can rupture, alsocausing bleeding into the brain. Hemorrhagic stroke in one part of thebrain can lead to ischemic stroke in another through shortage of bloodlost in the hemorrhagic stroke.

In patients with unruptured aneurysms, strokes that areprocedurally-related arise either as a result of emboli being dislodgedby the endovascular surgery (most common), or due to other complicationsof the endovascular surgery, such as perforation of the aneurysm(producing a hemorrhagic stroke) or inadvertent occlusion of a parentvessel due to mis-placement of endovascular coils or stents, orinadvertent occlusion of a parent vessel due to injury to the vesselthat results in thrombosis, dissection, or perforation.

In patients with ruptured aneurysms whose aneurysms are treated byendovascular means, strokes that are procedurally-related can arise forthe same reasons as in patients with unruptured aneurysms. However, suchpatients can also sustain additional ischemic or hemorrhagic braininjury because ruptured aneurysms are more fragile than unruptured ones,so the risk of the aneurysm rupturing before or during the procedure ishigher. Additional injury can result from increased intracranialpressure due to the original aneurysm rupture causing brain swelling(edema), or due to the intracerebral accumulation of the leaking blood,or both, or delayed ischemia due to the phenomenon of “vasospasm”. Insubarachnoid hemorrhage the risk of vasospasm is greatest between days5-12 after the aneurysm rupture, and is the result of vasoactivesubstances being released by the blood clot that surrounds the brainarteries. Vasospasm can be responsible for delayed ischemic strokes insuch patients. Damage can also arise from alterations in cerebral bloodflow due to loss of cerebral vascular autoregulation from a sudden riseof intracranial pressure immediately following the rupture.

V. Subjects Amenable to Treatment

The clinical trial provides evidence that a PSD-95 inhibitor iseffective in reducing infarcts and neurocognitive deficits in subjectsundergoing endovascular repair of both unruptured and rupturedaneurysms. Subjects with an unruptured aneurysm are predominantly atrisk of ischemic stroke or subarachnoid hemorrhage. Subjects withruptured aneurysms are also at risk of ischemic stroke but have addedrisk of hemorrhagic stroke, particularly from subarachnoid hemorrhage.Because of the added risk of hemorrhagic stroke, subjects with rupturedaneurysms have the greatest risk of death or debilitating injury asresult of the aneurysm. Surprisingly, the present data shows that thesepatients derive the greatest benefit from a PSD-95 inhibitor determinedby both pathology (number and volume of infarcts) and neurocognitivetesting. These results indicate not only that a PSD95 inhibitor can beused to treat ischemic or hemorrhagic stroke but provide evidence thatsuch an inhibitor can be used for treatment of subjects with hemorrhagesin or otherwise affecting the CNS, whether or not resulting from stroke.This is surprising because PSD-95 inhibitors have generally been thoughtto act though the reduction of ischemia, leading to better outcomes. InSAH, PSD-95 inhibitors such as Tat-NR2B9c were given within 72 hours ofrupture, when generally no ischemia is present, yet showed benefit.Ischemia in SAH patients generally occurs after vasospasm, and generallypresents between day 5 and day 12 post rupture. Tat-NR2M9c has a shorthalf life in plasma (about 20 minutes) and a short half-life in thebrain (about 5 hours), suggesting TAT-NR2B9c acts at least in partthrough a different mechanism than reducing ischemia to provide thebenefit observed in the examples below. However, practice of theinvention is not dependent on an understanding of mechanism.

The most common hemorrhages in or otherwise affecting the CNS arecerebral hemorrhage, intracerebral hemorrhage, intracranial hemorrhage(ICH) (each occurring inside the brain), and subdural and epiduralhemorrhages, and subarachnoid hemorrhage (SAH) (each occurring insidethe skull but outside the brain itself). These hemorrhages are referredto as being a hemorrhagic stroke if they occur spontaneously as whenaneurysm ruptures or a blood vessel leaks from hypertension, or drugssuch as anti-coagulants or cocaine, but are referred to simply ashemorrhage if they are from physical trauma, such as a fall, blow orshaken baby syndrome. The present methods are particularly suitable fortreatment of subarachnoid hemorrhage because this form of hemorrhage waspresent in the subjects deriving greatest benefit from the clinicaltrial.

Subjects amenable to treatment include subjects presenting with signs(s)and/or symptom(s) of ischemia or hemorrhage either in the CNS orimmediately proximate thereto as in the case of a subarachnoid, subduralor epidural hemorrhage or elsewhere in the body but still otherwiseaffecting the CNS as when affecting a blood vessel whose obstruction mayimpede blood flow through the brain, or in which hemorrhage may causeinjury through edema, pressure of accumulating blood or otherwise. Thesesubjects include subjects presenting with sign(s) and/or symptoms ofstroke, myocardial ischemia, pulmonary embolism, limb ischemia, renal,or retinal ischemia or hemorrhage in or proximate to the brain (e.g.,subarachnoid hemorrhage). Such subjects include subjects in which such acondition is suspected but other conditions cannot be excluded, as wellas subjects who have been diagnosed according to generally recognizedcriteria, e.g., DSM IV TR.

Subjects amenable to treatment also include subjects at risk of ischemiaor hemorrhage but in which onset of ischemia or hemorrhage has not yetoccurred. A subject is at risk if he or she has a higher risk ofdeveloping ischemia or hemorrhage than a control population. The controlpopulation may include one or more individuals selected at random fromthe general population (e.g., matched by age, gender, race and/orethnicity) who have not been diagnosed or have a family history of thedisorder. A subject can be considered at risk for a disorder if a “riskfactor” associated with that disorder is found to be associated withthat subject. A risk factor can include any activity, trait, event orproperty associated with a given disorder, for example, throughstatistical or epidemiological studies on a population of subjects. Asubject can thus be classified as being at risk for a disorder even ifstudies identifying the underlying risk factors did not include thesubject specifically. For example, a subject undergoing heart surgery isat risk of transient cerebral ischemic attack because the frequency oftransient cerebral ischemic attack is increased in a population ofsubjects who have undergone heart surgery as compared to a population ofsubjects who have not.

Subjects at risk of ischemia in or affecting the CNS include thoseundergoing a surgical procedure on the brain or CNS, such asendovascular surgery, clipping, stenting or microcathetherization. Suchsubjects also include those undergoing surgery elsewhere in the bodythat affects a blood vessel supplying the brain (that is connecting thebrain to the heart, for example, carotid arteries and jugular veins) oron an artery supplying blood to the retina, kidney, spinal cord orlimbs. Subjects at risk of hemorrhage affecting the CNS also includingthose undergoing a surgical procedure on the brain. Other subjects atrisk include those having had an injury to the head such as fall orblow, or have been subject to sudden changes in velocity, such as inshaken baby syndrome or a traffic accident. Other subjects at risk ofhemorrhage are those with hypertension, clotting disorders,arteriovenous malformation or aneurysm. A preferred class of subjectsare those undergoing endovascular surgery to treat a brain aneurysm withor without rupture.

VI. Combined Methods of Treatment

For ischemic indications, a PSD-95 inhibitor and a form of reperfusioncan be administered to a subject amenable to treatment (see US61/501117, filed Jun. 24 ,2011, incorporated by reference for allpurposes). The PSD-95 inhibitor and reperfusion can be administered ineither order or at the same time. Usually, the PSD-95 inhibitor andreperfusion are administered at the same, overlapping or proximate times(i.e., within a 15 minutes interval) or the PSD-95 inhibitor isadministered first.

For treatment of ischemias that cannot be predicted in advance, thePSD-95 inhibitor can be administered as soon as practical after onset ofischemia. For example, the PSD-95 inhibitor can be administered within aperiod of 0.5, 1, 2, 3, 4, 5 or 6, 12 or 24 hours or such other time asthere remains sufficient collateral circulation to maintain an ischemicpenumbra after onset of ischemia. For ischemias or hemorrhage that canbe predicted in advance, or are potential causes of symptoms, the PSD-95inhibitor can be administered before, concurrent with or after onset ofischemia. For example, for an ischemia or hemorrhage resulting fromsurgery, the PDS95 is sometimes routinely administered in a periodstarting 30 minutes before beginning surgery and ending one hour aftersurgery without regard to whether ischemia has or will develop. Becausethe PSD-95 inhibitor is free of serious side effects, it can beadministered when stroke, hemorrhage or other ischemic condition aresuspected without a diagnosis according to art-recognized criteriahaving been made. For example, the PSD-95 inhibitor can be administeredin an ambulance transporting a subject to a hospital. The PSD-95inhibitor can also be safely administered to a subject at risk of strokeor other ischemic condition or hemorrhagic condition before onset whomay or may not actually develop the condition. The PSD-95 inhibitor canalso be administered in the operating room or endovascular suite to ananesthetized patient on the presumptive diagnosis of ongoing orimpending brain ischemia or other injury.

Following, or sometimes before, administration of the PSD-95 inhibitor,a subject presenting with sign(s) and/or symptom(s) of ischemia can besubject to further diagnostic assessment to determine whether thesubject has ischemia within or otherwise affecting the CNS and determinewhether the subject has or is susceptible to hemorrhage. Mostparticularly in subjects presenting with symptoms of stroke or otheracute disorder affecting the CNS, testing attempts to distinguishwhether the subject has a hemorrhage. Diagnostic tests can include ascan of one or more organs, such as a CAT scan, MRI or PET imaging scan.The organ(s) scanned include any suspected as being the site of ischemia(e.g., brain, heart, limbs, spine, lungs, kidney, retina) as well as anyotherwise suspect of being the source of a hemorrhage. A scan of thebrain is the usual procedure for distinguishing between ischemic andhemorrhagic conditions. Diagnostic assessment can also include taking orreviewing a subject's medical history and performing other tests.Presence of any of the following factors alone or in combination can beused in assessing whether reperfusion therapy presents an unacceptablerisk: subject's symptoms are minor or rapidly improving, subject hadseizure at onset of stroke, subject has had another stroke or serioushead trauma within the past 3 months, subject had major surgery withinthe last 14 days, subject has known history of intracranial hemorrhage,subject has sustained systolic blood pressure >185 mmHg, subject hassustained diastolic blood pressure >110 mmHg, aggressive treatment isnecessary to lower the subject's blood pressure, subject has symptomssuggestive of subarachnoid hemorrhage, subject has had gastrointestinalor urinary tract hemorrhage within the last 21 days, subject has hadarterial puncture at noncompressible site within the last 7 days,subject has received heparin with the last 48 hours and has elevatedPTT, subject's prothrombin time (PT) is >15 seconds, subject's plateletcount is <100,000 μL. subject's serum glucose is <50 mg/dL or >400 mg/dLsubject is a hemophiliac or has other clotting deficiencies.

The further diagnostic investigation determines according to recognizedcriteria or at least with greater probability that before theinvestigation whether the subject has an ischemic condition, and whetherthe subject has a hemorrhage, has an unacceptable risk of hemorrhage oris otherwise excluded from receiving reperfusion therapy due tounacceptable risk of side effects. Subjects in which a diagnosis of anischemic conditions within or otherwise likely to affect the CNS isconfirmed who are without unacceptable risk of side effects can then besubject to reperfusion therapy. Preferably, reperfusion therapy isperformed as soon as practical after completion of any diagnosticprocedures. In some subjects, reperfusion therapy is commenced more than1, 2, 3, 4, 4.5, 5, 6, 7, 8, 10, 12, 15, 18, or 24 hr after onset ofischemia. In some subjects, reperfusion therapy is commenced 1-6, 1-12,1-18 or 1-24 hr after onset of ischemia. In some subjects, reperfusiontherapy is commenced outside the usual 3-4.5 hr window in whichreperfusion therapy has hitherto been considered to effective. Forexample in some subjects, reperfusion therapy is commenced more than 3hours or more than 4.5 hours after onset of ischemia and up to 24 or 48hours after onset of ischemia. In some subjects, reperfusion therapy iscommenced, after 5, 6, 7, 8, 9 or 10 hours and up to 24 or 48 hoursafter onset of ischemia. In some subjects, reperfusion therapy iscomments from 275-390 minutes after onset of ischemia.

The time of reperfusion can also be measured from the administration ofthe PSD-95 inhibitor. The interval can be, for example, 5 minutes to 24hours. The interval may be for example, 30 minutes to 6 hours or 1-3hours.

Subjects in which an ischemic condition is not confirmed or isconsidered unlikely are not usually administered reperfusion therapy,particularly pharmacological reperfusion therapy. Subjects in which anischemic condition is confirmed or considered likely but are consideredat unacceptable risk of side effects from pharmacological reperfusiontherapy are not administered pharmacological reperfusion therapy. Suchsubjects may have obtained benefit of the PSD-95 inhibitor but arespared the risk of unacceptable side effects from reperfusion therapy.

Both treatment with a PSD-95 inhibitor and reperfusion therapyindependently have ability to reduce infarction size and functionaldeficits due to ischemia. When used in combination, the reduction ininfarction size and/or functional deficits is preferably greater thanthat from use of either agent alone administered under a comparableregime other than for the combination. More preferably, the reduction ininfarction side and/or functional deficits is at least additive orpreferably more than additive of reductions achieved by the agents aloneunder a comparable regime except for the combination. In some regimes,the reperfusion therapy is effective in reducing infarction size and/orfunctional times at a time post onset of ischemia (e.g., more than 4.5hr) when it would be ineffective but for the concurrent or prioradministration of the PSD-95 inhibitor. Put another way, when a subjectis administered a PSD-95 inhibitor and reperfusion therapy, thereperfusion therapy is preferably at least as effective as it would beif administered at an earlier time without the PSD-95 inhibitor. Thus,the PSD-95 inhibitor effectively increases the efficacy of thereperfusion therapy by reducing one or more damaging effects of ischemiabefore or as reperfusion therapy takes effects. The PSD-95 inhibitor canthus compensate for delay in administering the reperfusion therapywhether the delay be from delay in the subject recognizing the danger ofhis or her initial symptoms delays in transporting a subject to ahospital or other medical institution or delays in performing diagnosticprocedures to establish presence of ischemia and/or absence ofhemorrhage or unacceptable risk thereof. Statistically significantcombined effects of PSD-95 inhibitor and reperfusion therapy includingadditive or synergistic effects can be demonstrated between populationsin a clinical trial or between populations of animal models inpreclinical work.

Subjects in which hemorrhage in or otherwise affecting the CNS isconfirmed, or cannot be excluded with acceptable assurance to thephysician are not usually administered pharmacological reperfusiontherapy but can be subject to other combination treatments, bothsurgical and pharmacological. Treatment with anti-PSD-95 inhibitors canalso take place in combination with other drugs, treatments orinterventions useful for indications associated with hemorrhages in orotherwise affecting the CNS, including SAH and ICH. Hemorrhages in orotherwise affecting the CNS can be treated by surgical intervention, anddepending on the diagnosis, therapeutics or treatments, such as forexample, antihypertensive medications, FactorVIIa or other clotting orcoagulation factors, mannitol or other drugs to raise intracranialpressure, acetaminophen or other NSAIDs to reduce headaches and avoidhyperthermia, frozen plasma, vitamin K, protamine, platelettransfusions, fosphenytoin or anticonvulsants if seizures are present orfor lobar hemorrhage, H2 antagonists or proton pump inhibitors forstress ulcer prophylaxis linked to ICH, or corticosteroids to reduceswelling. Because no safety or drug related interactions were observedin a human clinical trial of the PSD-95 inhibitor Tat-NR2B9c, suchcombination treatments are expected to be effective.

In other methods, in which a subject having or at risk of ischemia in orotherwise affecting the CNS, a subject is administered a PSD95 inhibitorwithout co-administration of other pharmacological treatment to treat oreffect prophylaxis of the ischemia. In some methods, such a subject isadministered a PSD95 inhibitor without administering otherpharmacological treatment to treat or effect prophylaxis of ischemia andwithout performing mechanical reperfusion therapy. In some methods, asubject having or at risk of a hemorrhage in or otherwise affecting theCNS is administered as PSD95 inhibitor without any other pharmacologicaltreatment to treat or effect prophylaxis of the hemorrhage.

VI. Effective Regimes of Administration

A PSD-95 inhibitor is administered in an amount, frequency and route ofadministration effective to reduce, inhibit or delay one or moredamaging effects of ischemia or hemorrhage on the CNS and preferablypain associated with either the ischemia or hemorrhage or surgicaltreatment thereof. Unless otherwise indicated dosages for inhibitorsthat are chimeric agents including a pharmacologic agent linked to aninternalization peptide refer to the whole agent rather than just thepharmacological agent component of the chimeric agent. An effectiveamount means an amount of agent sufficient significantly to reduce,inhibit or delay onset or more damaging effects of ischemia orhemorrhage and preferably pain as well in a population of subjects (oranimal models) suffering from the disease treated with an agent of theinvention relative to the damage in a control population of subjects (oranimal models) suffering from that disease or condition who are nottreated with the agent. The control population can be contemporaneouslytreated with a placebo or can be a historical control. The amount isalso considered effective if an individual treated subject achieves anoutcome more favorable than the mean outcome in a control population ofcomparable subjects not treated by methods of the invention. Aneffective regime involves the administration of an effective dose at afrequency and route of administration needed to achieve the intendedpurpose.

The outcome of treating stroke or hemorrhage affecting the CNS can bedetermined by infarction volume, number of infarctions, or disabilityindex. A regime can be recognized as effective if an individual treatedsubject shows a disability of two or less on the Rankin scale and 75 ormore on the Barthel scale, see Lees et a., N. Engl. J. Med. 354:588-600(2006) or if a population of treated subjects shows a significantlyimproved (i.e., less disability) distribution of scores on any stroke,disability or other appropriate scale (e.g., Barthel, Rankin, NIH StrokeScale) than a comparable untreated population, or if a population oftreated subjects shows significantly reduced infarction size or numbercompared with a comparable untreated population. A single dose of agentis usually sufficient for treatment of stroke.

Infarctions in a clinical trial or individual patient are preferablyassessed by MRI, particularly FLAIR (fluid attenuated inversionrecovery) and/or DWI (diffusion weighted imaging). FLAIR is moresensitive but DWI is more specific for new infarctions. Identificationof infarctions present at the same spatial location by both FLAIR andDWI provide a sensitive and selective detection of new infarctionsresulting from a current episode of stroke or hemorrhage. Other MRIsequences can be used alone or in combination with DWI and/or FLAIR.

Depending on the agent, administration can be parenteral, intravenous,nasal, oral, subcutaneous, intra-arterial, intracranial, intrathecal,intraperitoneal, topical, intranasal or intramuscular. Intravenousadministration is preferred for peptide agents.

For chimeric agents including an internalization peptide, particularly aHIV tat peptide comprising the amino acid sequence, administration ofthe agent may or may not be combined with an anti-inflammatory agent toreduce release or histamine and its downstream effects associated withhigh levels of the internalization peptide. Preferred agents forco-administration are inhibitors of mast cell degranulation, such ascromolyn or lodoxamide or any others listed herein. Anti-histamines orcorticosteroids can also be used, particularly in combinations or higherdosages (see WO2009/076105, and WO2010/14474261).

For administration to humans, a preferred dose of the chimeric agentTat-NR2B9c is 2-3 mg/kg and more preferably 2.6 mg/kg. Indicated dosagesshould be understood as including the margin of error inherent in theaccuracy with which dosages can be measured in a typical hospitalsetting. The dose is preferred because it is the maximum dose with whichthe agent can be administered without release of significant amounts ofhistamine and the ensuing sequelae in most subjects. Although release ofhistamine at higher dosages can be controlled by co-administration of ananti-inflammatory as discussed above and in any event usuallyspontaneously resolves without adverse events, it is best avoided bykeeping the dose below 3 mg/kg and preferably at 2-3 mg/kg, morepreferably 2.6 mg/kg. Another preferred dose level is 1-3 mg/kg, e.g.,1.5 mg/kg. Such amounts can be for single dose administration, i.e., onedose per episode of disease, or multiple dose administration.

The dosages indicated above are for the chimeric agent Tat-NR2B9c(YGRKKRRQRRRKLSSIESDV; SEQ ID NO:6). Equivalent dosages for other agentsto achieve the same effect can be determined by several approaches. Forclose variants of that agent in which one or a few amino acids aresubstituted, inserted or deleted and the molecular weight remains thesame within about +/−25%, the above dosages are still a good guide.However, in general, for other agents, equivalent dosages can varydepending on the molecular weight of the agent with and withoutinternalization peptide if present, its Kd for its target, and itspharmacokinetic and pharmacodynamic parameters. For some agents,equivalent dosages can be calculated so as to deliver an equimolaramount of the pharmacological agent. For other agent, further adjustmentis made to account for differences in Kd or pharmacokinetic orpharmacodynamic parameters. For some agents, equivalent dosages aredetermined empirically from the dose achieved to reach the same endpointin an animal model or a clinical trial.

Peptide agents, such as Tat-NR2B9c are preferably delivered by infusioninto a blood vessel, more preferably by intravenous infusion. For thechimeric agent Tat-NR2B9c, a preferred infusion time providing a balancebetween these considerations is 5-15 minutes and more preferably 10 min.Indicated times should be understand as including a marking of error of+/−10%. Infusion times do not include any extra time for a wash outdiffusion to wash out any remaining droplets from an initial diffusionthat has otherwise proceeded to completion. The infusion times forTat-NR2B9c can also serve as a guide for other pharmacological agents,optionally linked to internalization peptides, particularly closevariants of Tat-NR2B9c, as discussed above.

Multi-dose regimes of Tat-NR2B9c or other PSD95 inhibitor can also beused. For example, multi-dose regimes can be used for treatingsubarachnoid hemorrhage or other hemorrhages of the CNS. Multi-doseregimes can involve administering a PSD95 inhibitor once or twice a day(or more) for up to 12 days starting on the day of hemorrhage. In onepreferred regime, the inhibitor is administered once a day for at leastthree days. In another preferred regime, the inhibitor is administeredtwice a day for at least two days. In some regimes, at least one dose isadministered within 4 days of rupture (i.e., on or before day 4 withrupture being on day 1). In some regimes, a dose is administered on day5 or later after rupture. In some regimes, a dose is administered on anyor all of days 1-4 and another dose on any or all of days 5-12. In someregimes, one dose is administered within days 1-4 and other dose withindays 5-12. Doses can be e.g.,1-3 mg/kg preferably 2-3 mg/kg or 2.6mg/kg.

The PSD-95 inhibitor can be administered in the form of a pharmaceuticalcomposition. Pharmaceutical compositions are typically manufacturedunder GMP conditions. Pharmaceutical compositions for parenteraladministration are preferentially sterile (e.g., filter sterilization ofpeptide) and free of pyrogens. Pharmaceutical compositions can beprovided in unit dosage form (i.e., the dosage for a singleadministration). Pharmaceutical compositions can be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries that facilitate processingof chimeric agents into preparations which can be used pharmaceutically.Proper formulation is dependent on the route of administration chosen.

An exemplary formulation of the chimeric agent Tat-NR2B9c contains thepeptide in normal saline (0.8-1.0% and preferably 0.9% saline) orphosphate buffered saline at a concentration of 10-30 mg/ml, for example16-20 or 18 mg/ml. When stored frozen, such a composition is stable(insignificant degradation or aggregation of the peptide) for a periodof two or more years. Although additional excipients can be added,normal saline or phosphate buffered saline without such excipients issufficient to obtain this stability. For use such a composition isthawed and diluted into a larger volume of normal saline for infusioninto a blood vessel.

Many examples of pharmacological agent for reperfusion are in clinicaluse. Such agents can be used in the present combination methods inaccordance with their conventional formulations, doses, routes ofadministration, and frequency of administration (see Physician's DeskReference and applicable package inserts). Likewise, mechanical methodsof reperfusion can be employed in accordance with conventional practice.

EXAMPLES Example 1 Neuroprotection by TAT-NR2B9C after Ischemic Strokeor Brain Hemorrhage Study Design

We conducted a randomized, double blind, placebo-controlled trial inwhich patients were enrolled from September 2008 through March 2011 at14 hospitals in Canada and the USA. The trial was approved by local andnational institutional review boards, and informed consent was obtainedfrom the patients or from legally acceptable surrogates. The inclusionand exclusion criteria are summarized in Table 4. All patients receivedtreatment of their aneurysm and, in the case of a ruptured aneurysm, oftheir subarachnoid haemorrhage, in accordance with local institutionaltreatment practices.

TABLE 4 Major Inclusion and Exclusion Criteria Inclusion CriteriaRuptured or unruptured brain aneurysm deemed suitable for repair byneuroendovascular techniques involving detachable platinum coils, stent-assisted coiling, flow-diverting stents, balloon-assisted coiling,covered stents, neck-bridge devices, or any other adjunctive devicesRecoiling or re-treatment of previously treated aneurysm If rupturedaneurysm Endovascular repair must take place within 72 hours of theictal haemorrhage Subject should be WFNS Grade I-III Body weight lessthan or equal to 180 kg Normal or abnormal but not clinicallysignificant findings in the non-neurological physical exam, 12-lead ECGand vital signs Absence of ongoing ischemic symptoms such as TIAs, minorstrokes, stroke-in-evolution, or clinical evidence of cerebral vasospasmwithin 2 weeks prior to randomization Brain MRI within 2 weeks prior tothe endovascular repair Male or female with a minimum age of 18 yearsFemales of childbearing potential must have a negative pregnancy testand be willing to use birth control for 3 months after completion ofdosing Non-surgically sterile males or males with partners ofchildbearing potential must be willing to use condoms with spermicidefor 3 months Signed informed consent and availability of the subject forthe entire study period Exclusion Criteria Dissecting or mycotic brainaneurysm Planned endovascular vessel sacrifice as the primary modalityfor aneurysm treatment Known history of life-threatening allergicreaction to any medication Chronic renal disease defined as a baselineserum creatinine >150 μmol/L Women who are breastfeeding Any clinicallysignificant psychiatric or psychological disease which would precludesubject from completing protocol Pre-morbid (estimated) modified Rankinscale score of >2 Previous serious traumatic brain injury that wouldpreclude the subject from completing the protocol or preclude MRIanalysis of small strokes Subjects who are unable to have an MRI scanfor any reason Participation in another clinical trial with aninvestigational drug within 30 days of this study including ENACT orprior receipt of TAT-NR2B9C Any other medical condition that would putthe subject at excessive risk of participation in the study, or anexpected life expectancy <1 year or that would result in inability tocollect clinical outcomes at Day 30

Clinical and MRI Assessments

All clinical and MRI assessments were performed by individuals who wereunaware of the treatment assignment. Each enrolled patient underwent anMRI scan on a (minimum) 1.5T scanner within 2 weeks prior to theendovascular procedure and at 24-96 hours thereafter. Each scan includedthe following minimum sequences: an axial FLAIR: 3 mm, no gap, an axialDWI: 3 mm, no gap and an axial 3D T1 weighted gradient echo sequence 2.0mm (e.g. FSPGR on GE machine).

Patients were assessed at the time of enrolment, post-procedure, andover a 30 day study period as detailed in Table 5. Initial assessmentsincluded a physical examination, neuroimaging, baseline scoring on theNational Institutes of Health Stroke Scale (NIHSS) and the modifiedRankin scale (mRS) and a neurocognitive battery (detailed in Table 6).For patients with ruptured aneurysms, baseline neurocognitive testingwas omitted, the subarachnoid haemorrhage (SAH) was assigned a Fishergrade and its clinical severity was scored according to the WorldFederation of Neurosurgical Societies (WFNS) grading system. The NIHSSis a 15-item scale that measures the level of neurologic impairment.Scores range from 0 to 42, with a higher score indicating greater strokeseverity. The mRS is a measure of disability that ranges from 0 (nosymptoms at all) to 6 (death); a score of 5 indicates severe disability(the patient is bedridden and incontinent and requires constant nursingcare and attention). The WFNS system (Grades 1-5) rates the clinicalcondition of a SAH patient according to the Glascow Coma Score (GCS),where Grade 1 is normal, and Grade 5 is GCS <7. The Fisher gradingsystem classifies the appearance of subarachnoid hemorrhage on a CT inorder to predict the risk of cerebral vasospasm. The examiners weretrained and certified in the use of all scales. Clinical assessmentspost-procedure and drug infusion were focused primarily oncardiorespiratory safety, neurological function, and neurocognition. Forpatients with ruptured aneurysms, neurocognitive studies were onlyperformed on day 30.

TABLE 5 Clinical, biochemical and MRI assessment flow chart. StudyPeriod Enrolment (Within 14 Dosing Days 2-4 End of Study Procedure d ofDay 1) Day 1 (24-96 hours) (Day 30 ± 7 d) Informed Consent X Medical andSurgical History X Demographics X WFNS and Fisher Grade X (rupturedaneurysm patients only) Modified Rankin Scale (mRS) X X X NIH StrokeScale (NIHSS) X X¹ X X Cognitive Battery² X X X Physical/NeurologicalExamination X X³ X Vital Signs Measurement⁴ X⁵ X⁶ X⁷ X⁷ Height X WeightX X⁸ ECG X X⁹ X¹⁰ Histamine Sample Collection¹¹ X Bioanalytical SampleCollection¹² X Biochemistry, Hematology, Urinalysis X X¹³ X¹³ X Serumβ-hCG Testing¹⁴ X X X Urine Pregnancy Testing¹⁴ X Plasma sample forstorage (for X¹⁵ X possible immunogenicity testing) MRI Imaging: DWI andFLAIR sequences X X ¹1-4 hours post exit from the angiography suite²Enrolment and Days 2-4 assessment omitted for patients with a rupturedaneurysm (performed on Day 30 only). ³Postdose. ⁴Cardiac monitoring wasperformed on Day 1 from 0 to 2 hours post-dose. ⁵Blood pressure, heartrate, temperature, SaO₂. ⁶Neurovital signs performed at least every 4hours until 24 hours after dosing and then as determined by the treatingphysician(s). BP, HR, and O₂ saturation to were measured pre-procedure(i.e., pre-anesthesia induction) and post-procedure (immediatelypre-dose). BP and HR: 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 16, 20 and 24 hourspost-dose. Temperature measured once 12-24 hours post-dose. O₂saturation: 0.5, 1, 2, 3, 4, 5, 6 post-dose, but could be discontinuedafter termination of, and recovery from, anesthesia, and is was notrequired beyond 6 hours post-dose. ⁷Blood pressure, heart rate,temperature. ⁸Weight to be measured for all patients at Enrolment andDay 2-4. In the case of ruptured aneurysm patients whose weight has tobe estimated at Enrolment (following clinical practice), actual weightis required to be measured by Day 2-4. ⁹12-lead ECG pre-dose, 4-6 hours,and 12 hours post-dose. If Enrolment and Day 1 occur on the same day,one 12-lead ECG may be performed to serve as both the Enrolment and Day1 pre-dose ECG. (Minimum of) 3-lead ECG monitoring will be performed bythe anaesthesiologist and/or post-operative recovery room staff fromonset of dosing until at least 2 hours post-dose. ¹⁰12-lead ECG 24 hourspost-dose ¹¹Two samples taken at predose and at 10 minutes after thetermination of the study drug infusion. ¹²Two consecutive samples for PKanalysis, taken between 5 and 10 minutes after the start of the studydrug infusion. ¹³As per institutional practice patterns in the treatmentof patients with brain aneurysms post-endovascular treatment. ¹⁴Forwomen of childbearing potential only; either urine or serum testing canbe performed predose on Day 1. See section 8.5.2 for further details.¹⁵Predose

TABLE 6 Day 30 Neurocognitive Outcomes Placebo TAT-NR2B9C Mean ± SD Mean± SD P Test Direction* N= N= Value All Randomized Subjects Day 30 TrailsA ⬇ 48.10 ± 34.20 37.55 ± 18.50 0.083 Trails B ⬇ 115.27 ± 77.80  101.12± 75.10  0.402 Phonemic Verbal Fluency ⬆ 36.20 ± 13.10 34.80 ± 12.000.469 Semantic Verbal Fluency ⬆ 17.78 ± 5.48  16.36 ± 4.91  0.075 DigitSymbol ⬆ 65.04 ± 22.40 60.58 ± 21.10 0.186 HVLT-R Total Recall ⬆ 23.22 ±5.24  22.70 ± 6.26  0.555 HVLT-R Delayed Recall ⬆ 8.09 ± 2.59 7.37 ±3.25 0.109 HVLT-R Retention % ⬆ 85.10 ± 18.70 76.61 ± 26.90 0.017 HVLT-RRDI ⬆ 10.02 ± 1.69  9.63 ± 2.61 0.240 MMSE ⬆ 28.50 ± 1.68  28.50 ± 2.62 1.000 NPI-Q Severity Score ⬇ 3.48 ± 4.73 1.80 ± 2.32 0.014 NPI-QDistress Score ⬇ 3.23 ± 5.23 1.56 ± 2.49 0.026 CES-D ⬇ 11.62 ± 11.2010.75 ± 8.11  0.556 Unruptured Subjects Day 30 Trails A ⬇ 46.22 ± 32.8036.03 ± 19.10 0.115 Trails B ⬇ 107.86 ± 72.36  96.601 ± 73.48  0.517Phonemic Verbal Fluency ⬆ 37.67 ± 12.00 34.68 ± 12.40 0.146 SemanticVerbal Fluency ⬆ 18.32 ± 4.58  16.13 ± 4.72  0.006 Digit Symbol ⬆ 66.88± 21.70 62.12 ± 21.10 0.196 HVLT-R Total Recall ⬆ 23.70 ± 4.96  23.12 ±6.00  0.528 HVLT-R Delayed Recall ⬆ 8.33 ± 2.54 7.43 ± 3.15 0.063 HVLT-RRetention % ⬆ 86.71 ± 18.20 76.17 ± 25.7  0.005 HVLT-R RDI ⬆ 10.01 ±1.75  9.68 ± 2.64 0.370 MMSE ⬆ 28.50 ± 1.73  28.71 ± 2.33  0.661 NPI-QSeverity Score ⬇ 2.95 ± 4.19 1.57 ± 2.02 0.046 NPI-Q Distress Score ⬇2.62 ± 4.39 1.31 ± 2.07 0.068 CES-D ⬇ 11.35 ± 11.00 9.57 ± 7.81 0.272Ruptured Subjects Day 30 Trails A ⬇ 61.60 ± 45.50 45.14 ± 13.70 0.3807Trails B ⬇ 168.60 ± 103.00 123.71 ± 85.10  0.4272 Phonemic VerbalFluency ⬆ 27.92 ± 16.30 35.29 ± 10.90 0.1498 Semantic Verbal Fluency ⬆14.77 ± 8.68  17.22 ± 5.63  0.3473 Digit Symbol ⬆ 54.85 ± 24.10 54.94 ±20.70 0.9904 HVLT-R Total Recall ⬆ 20.54 ± 6.13  21.11 ± 7.13  0.8169HVLT-R Delayed Recall ⬆ 6.77 ± 2.55 7.17 ± 3.67 0.7395 HVLT-R Retention% ⬆ 76.05 ± 19.5  78.25 ± 31.9  0.8269 HVLT-R RDI ⬆ 10.08 ± 1.32  9.44 ±2.55 0.4209 MMSE ⬆ 28.50 ± 1.29  27.43 ± 3.78  0.6033 NPI-Q SeverityScore ⬇ 5.08 ± 5.99 2.53 ± 3.07 0.161 NPI-Q Distress Score ⬇ 5.08 ± 7.092.40 ± 3.50 0.2069 CES-D ⬇ 13.15 ± 12.2  15.28 ± 7.80  0.5591 *⬆ Thehigher the score the better the test performance ⬇ The lower the scorethe better the test performance

Study Interventions

Patients were randomly assigned by a computer-generated coding system toreceive an intravenous infusion of either TAT-NR2B9C or Placebo. Thestudy drug was supplied as a drug vial containing 20 mg/nnl TAT-NR2B9C,which was dosed at 2.6 mg/kg by dilution in 100cc normal saline andinfused intravenously over 10 minutes. Infusion, performed byindividuals blinded to the treatment assignment, began once the treatingneurointerventionalist deemed the aneurysm repair to be completed butprior to termination of anesthesia. The rationale for this timing wasthat the purpose of ENACT was to test whether neuroprotection in humansis feasible after a stroke has occurred, and not just in a pre-treatmentparadigm.

Assessment of Stroke Number and Volume

New ischemic lesions were defined as new hyperintense signals on the day2-4 DWI MRI (FIGS. 1A-C; yellow arrows; termed DWI lesions). The volumeof new DWI lesions was determined from the surface area of regions ofinterest (ROIs) traced around each DWI lesion (FIGS. 1A-C), multipliedby the slice thickness (3 mm). Lesions on FLAIR imaging were deemed tobe new if they fell within the location of a new DWI lesion and did notpre-exist on the enrolment MRI (FIGS. 1A-C). The volumes of new FLAIRlesions were determined similarly to that of new DWI lesions. Allcalculations were performed using Osirix software (v.3.9.2, 32 bitversion).

Outcome Measures

The main purpose of ENACT was to test the neuroprotection hypothesis,Thus the effect of TAT-NR2B9C on the number and volume of detectable newlesions was of primary interest. However, the ENACT paradigm has neverbeen previously conducted, and the MRI assessments provided 4 measuresof equal interest (DWI lesion number, DWI lesion volume, FLAIR lesionnumber and FLAIR lesion volume). We selected as the primary efficacyoutcome measure the ability of a single intravenous dose of TAT-NR2B9Cto reduce the volume of embolic strokes as measured by DWI and FLAIR MRIImaging at 24-96 hours post-procedure. Another objective of primaryinterest was to determine the safety and tolerability of TAT-NR2B9C inthe present patient population. Secondary outcomes included the efficacyof TAT-NR2B9C in reducing the number of embolic strokes, efficacy inpatients sustaining small strokes (<10cc's), in reducingprocedurally-induced cognitive impairment at the day 30 follow-up, inreducing the frequency of large (>10cc strokes), and in improvingoutcome (new strokes, neurological and neurocognitive) in the subgroupsof patients with ruptured and unruptured aneurysms.

Statistical Analysis

Data were analyzed according to the modified intent-to-treat (mITT)principle; ITT; and per protocol populations. Under the mITT principle,the evaluable sample includes all subjects who are randomized andreceive any amount of study drug. In the case of missing data onclinical outcome among patients known to be alive, the worst possibleoutcome score was assigned. P values are presented unadjusted and, whereindicated, adjusted for variables that impact the outcome measure.

Results Study Patients

The disposition of all study patients is provided in FIG. 2. BetweenSeptember, 2008, and March 2011, 212 patients from 11 Canadian and 3 USAsites were screened in accordance with the inclusion/exclusion criteriaof the Trial (Table 4). 15 did not meet the criteria and were notrandomized. There were 12 randomization failures (Patient randomized,but did not receive study drug): 5 due to inability to carry out theendovascular aneurysm repair, 3 due to inability to obtain apre-procedure MRI (planned on the same day as the procedure), 2 due to apre-procedure ECG showing a QTc interval >450 ms (an exclusioncriterion), 1 due to a fatal aneurysm rupture during the procedure andprior to drug infusion and 1 due to refusal by the anesthesiologist toadminister study drug in a patient with severe chronic obstructivepulmonary disease. A total of 185 patients were thus randomized andreceived study drug (the mITT population), such that 92 patients wererandomly assigned to receive TAT-NR2B9C and 93 patients were assigned toreceive placebo (FIG. 2). In the TAT-NR2B9C group, 1 patient died priorto the day 2-4 MRI scan and 2 refused to attend their day 30 follow up.In the placebo group, 2 subjects died after the day 2-4 MRI scan, and athird did not attend the day 30 follow up (FIG. 2).

Baseline demographic and clinical characteristics of the two groups weresimilar (Table 7). The endovascular procedures lasted, on average, about2 hours (Table 7). About half of the aneurysm repairs could be performedusing only detachable platinum coils, whereas in the rest, additionalassistive techniques or devices were used comprising of balloon orstent-assisted coiling or repair using flow-diverting stents. There wereno differences in the baseline and clinical characteristics between theTAT-NR2B9C and Placebo groups in the subgroups of patients with rupturedor unruptured aneurysms, except for a higher incidence of hypertensionin patients with ruptured aneurysms treated with placebo (Table 7).

TABLE 7 Demographic and Baseline Characteristics of the Patients AllRandomized Unuptured Aneurysm Ruptured Aneurysm Study Group Study GroupStudy Group Placebo NA-1 Placebo NA-1 Placebo NA-1 Characteristic (N =93) (N = 92) P * (N = 74) (N = 74) P * (N = 19) (N = 18) P * Age (yr)56.05 ± 10.3    58 ± 11.05 0.21 56.48 ± 19.7  58.78 ± 9.7  0.17 54.47 ±9.12   55.0 ± 15.44 0.90 Male sex (%) 26.40% 30.40% 0.63 20.73% 31.08%1.00 15.79% 27.78% 0.45 Weight (Kg) 76.4 ± 16.5 75.0 ± 20.1 0.62 77.4 ±17.2 75.6 ± 20.0 0.55 72.6 ± 13.1 72.8 ± 25.2 0.97 Syst. pressure (mmHg) 130.5 ± 14.4  130.2 ± 16.2  0.87 130.9 ± 14.1  7 0.95  129 ± 16.1 126 ± 14.2 0.61 Dias. Pressure (mm Hg) 76.0 ± 10.0 75.8 ± 12.1 0.9176.6 ± 9.00 77.9 ± 11.2 0.46 73.6 ± 13.1 67.3 ± 12.2 0.15 Diabetes (%) 6.50%  5.40% 0.77  6.76%  5.41% 1.00  5.26%  5.56% 1.00 Hypertension(%) 50.50% 42.40% 0.8 45.95% 45.95% 1.00 68.43% 27.78% 0.02Hyperlipidemia (%) 10.80% 11.00% 0.8  9.46% 12.16% 0.79 15.79% 11.11%1.00 Smoking status (%) 0.06 0.19 0.19 No 36.60% 26.10% 33.78% 25.68%47.37% 27.78% Past 26.90% 43.50% 32.43% 47.30%  5.26% 27.78% Current36.60% 30.40% 31/78% 27.03% 47.37% 44.44% NIHSS score† Mean 0.45 ± 2.150.22 ± 0.53 0.31 0.18 ± 0.65 0.19 ± 0.51 0.89 1.93 ± 5.09 0.35 ± 0.610.21 Median 0 0 0.22 0 0 0.85 0 0 0.11 mRS Mean 0.28 ± 0.71 0.17 ± 0.410.22 0.24 ± 0.64 0.16 ± 0.41 0.36 0.42 ± 0.96 0.22 ± 0.43 0.43 Median 00 0.16 0 0 0.12 0 0 1.00 Ruptured Aneurysm (N) 20.40% 19.60% 0.88Procedure duration (h)   2.07   2.06 0.9 2.01 ± 1.10 2.03 ± 0.82 0.912.29 ± 1.00 2.13 ± 0.57 0.56 Assistive device (%)   52%   54% 0.7150.00% 59.46% 0.32 57.89% 33.33% 0.19 balloon   25%   29% stent   19%  18% pipeline stent    8%    7% Concom. Antiplatelet 41.00% 35.00% 0.4233.78% 48.55% 0.10 42.11% 11.11% 0.06 * P values were obtained using at-test or a Fisher's exact test. * Scores on the National Institutes ofHealth Stroke Scale (NIHSS) range from 0 to 42, with higher valuesreflecting more severe neurologic impairment (<5 mild impairment; ≥25,very severe impairment).

MRI Outcomes

Of 185 randomized subjects, 184 completed the post-procedure (day 2-4)MRI scan. Patients who were treated with TAT-NR2B9C after theirendovascular procedure exhibited a 43% reduction in the number of newischemic lesions as detected by DWI MRI (Table 8; p=0.005). This wasalso reflected in the FLAIR MRI scans, in which treatment withTAT-NR2B9C reduced the number of new ischemic lesions by 39% (Table 8;p=0.026). The median stroke volumes as measured by FLAIR and DWI MRIwere also reduced in patients treated with TAT-NR2B9C (Table 8; p<0.001for both DWI and FLAIR volumes [using a Rank-Ordered Logistic Regressionin Stata]). However, due to the skewed and widely dispersed distributionof the volumes data (Table 8), mean infarct volumes were significantlydistorted by patients who exhibited large strokes (defined as >10cc's).Specifically, two patients assigned to the TAT-NR2B9C group sufferedfrom complications during the aneurysm repair procedure that causedlarge strokes (10.7cc and 49.2 cc). This produced non-significantoverall differences in mean stroke volumes between the treatment groups.

TABLE 8 MRI Outcomes All Patients Study Group Placebo TAT-NR2B9C P Value(N = 93) (N = 91) P Value P Value MRI Parameter Mean Median Mean MedianUnadj* Adj** Number of DWI Lesions 7.28 ± 12.61 2 4.13 ± 6.81 2 0.0180.005 Number of FLAIR Lesions 4.83 ± 7.69  2 2.96 ± 4.42 1 0.048 0.026Volume of DWI Lesions 645 ± 1382 123.9  966 ± 5266 59.4 0.306 0.120(mm3) Volume of FLAIR Lesions 477 ± 1611 44.9  915 ± 5598 29.1 0.4450.236 (mm3) Patients with Ruptured Aneurysms Placebo TAT-NR2B9C (N = 19)(N = 18) P Value P Value Mean Median Mean Median Unadj* Adj** Number ofDWI Lesions  9.47 ± 11.59 4 3.39 ± 5.93 1 0.027 Number of FLAIR Lesions6.58 ± 7.48 4 2.39 ± 4.67 0 0.046 Volume of DWI Lesions 1373 ± 2267164.6 277 ± 528 28.7 0.015 Volume of FLAIR Lesions 1575 ± 3229 86.8 205± 495 0 0.023 Patients with Unruptured Aneurysms Placebo TAT-NR2B9C (N =74) (N = 73) P Value P Value Mean Median Mean Median Unadj* Adj** Numberof DWI Lesions  6.72 ± 12.88 2 4.32 ± 7.03 2 0.108 0.019 Number of FLAIRLesions 4.38 ± 7.64 2 3.10 ± 4.39 1 0.220 0.084 Volume of DWI Lesions459 ± 983 109.1 1137 ± 5870 72.2 0.933 0.471 Volume of FLAIR Lesions 195± 553 41.4 1083 ± 6215 33.2 0.617 0.896 Patients with Strokes <10 cc's†Placebo TAT-NR2B9C (N = 93) (N = 89) P Value P Value Mean Median MeanMedian Unadj* Adj** Number of DWI Lesions 7.28 ± 12.61 2 3.91 ± 6.72 10.010 0.002 Number of FLAIR Lesions 4.83 ± 7.69  2 2.75 ± 4.25 1 0.0240.012 Volume of DWI Lesions 645 ± 1382 123.9 315 ± 646 51.5 0.054 0.009Volume of FLAIR Lesions 477 ± 1611 44.9 183 ± 506 25.2 0.061 0.014Patients w Strokes <10 cc and unrupt. aneurysms Placebo TAT-NR2B9C (N =74) (N = 71) P Value P Value Mean Median Mean Median Unadj* Adj** Numberof DWI Lesions  6.72 ± 12.88 2 4.04 ± 6.94 2 0.069 0.010 Number of FLAIRLesions 4.38 ± 7.64 2 2.84 ± 4.17 1 0.131 0.045 Volume of DWI Lesions459 ± 983 109.1 325 ± 675 70.4 0.441 0.088 Volume of FLAIR Lesions 195 ±553 41.4 178 ± 511 30.2 0.581 0.183 Plus-minus values are means ± SD; *Pvalues reflect a test of the differences between the means. **Adjusted Pvalues represent the effect of treatment, adjusted for the variablesAge, Aneurysm Rupture, the use of Adjunctive Devices, procedure durationand the use of Antiplatelet Agents (ASA or Plavix).; †As defined by DWIvolume; ‡ Adjusted values not calculated due to small numbers ofpatients

Among the patients randomized in ENACT, 37 presented with a SAH due to aruptured aneurysm. This subgroup was preselected for a subgroup analysisdue to the possibility that the SAH might impact on the likelihood ofsuffering new ischemic lesions. Additionally, such patients might suffermore severe neurological and neurocognitive deficits due to the SAH thanpatients who undergo elective procedures for unruptured aneurysms. Inthis patient subgroup, treatment with TAT-NR2B9C reduced the number ofnew ischemic lesions detected by either DWI MRI or FLAIR MRI by 64%(p=0.027 and p=0.046 for DWI and FLAIR, respectively; Table 8).Moreover, the patients treated with TAT-NR2B9C exhibited an 80%reduction in new infarct volumes as defined by DWI MRI (p=0.015), and an87% reduction of new infarct volumes as defined by FLAIR MRI (p=0.023).In the subgroup of patients with unruptured aneurysms (n=147), theeffect of treatment with TAT-NR2B9C on the MRI outcomes trended in thesame directions as the overall patient cohort (Table 8).

An additional subgroup analysis was performed on patients without largestrokes (182 of the 184 patients). The rationale for this analysis wasthat the ENACT paradigm was designed to examine the effect of treatmenton small embolic strokes, similar to those modelled in the non-humanprimate experiments where in new ischemic lesions were consistentlysmall. However, in such a paradigm, the arithmetic means of the infarctvolumes became subject to distortion by infarcts that far exceed themedian volumes observed. In patients with strokes under 10cc's byvolume, treatment with TAT-NR2B9C reduced the numbers and volumes of newischemic lesions as detected by DWI and by FLAIR MRI (Table 8),consistent with a neuroprotective effect of TAT-NR2B9C in small,procedurally-induced strokes. Thus, the MRI methods described herein canalso be useful for screening the effects of other neuroprotective drugsin ischemic stroke, hemorrhagic stroke, and SAH.

A secondary objective in ENACT was to determine whether TAT-NR2B9Ctreatment reduces the frequency of large (>10cc) strokes. However, the 2occurrences of large strokes were insufficient to warrant statisticalanalysis.

Neurological Outcomes

Patients enrolled in ENACT either underwent elective aneurysm repair orexperienced a SAH with WFNS scores of I-III (Glascow Coma Score 13-15).Consequently, they exhibited a low level of neurological disability atthe time of the endovascular procedure (median NIHSS and mRS of 0; Table7). On day 30 Post-procedure, 93.5% of all patients who were treatedwith TAT-NR2B9C and 89.2% of patients treated with Placebo hadfavourable NIHSS scores (NIHSS 0-1, p=0.434; Table 9). mRS scores werefavorable (0-2) in 93.5% in each of the TAT-NR2B9C and placebo groups(Table 9). However, in subjects enrolled with ruptured aneurysms, 100%of those treated with TAT-NR2B9C had favorable NIHSS 30 dayspost-procedure, as compared with 68.4% of patients treated with placebo(p=0.020; Table 4). SAH patients treated with TAT-NR2B9C also trended tohaving more favorable mRS scores as compared with those treated withplacebo (94.4% vs 73.7% in TAT-NR2B9C and placebo, respectively;p=0.180; Table 9). The patients who sustained large strokes (>10cc) dueto procedural complications were in the TAT-NR2B9C group. On day 30, onehad a NIHSS of 2 and mRS of 2, and the other an NIHSS of 2 and mRS of 1.

TABLE 9 Neurological Outcomes Study Group Placebo no**. (%) TAT-NR2B9C Pvalue All Patients (N = 93) (N = 92) NIHSS of 0 or 1 83 (89.2%) 86(93.5%) 0.434 mRS score of 0-2 87 (93.5%) 86 (93.5%) 1.000 Patients with(N = 74) (N = 74) Unruptured Aneurysms NIHSS of 0 or 1 70 (94.6%) 68(91.9%) 0.745 mRS score of 0-2 73 (98.6%) 69 (93.2%) 0.363 Patients with(N = 19) (N = 18) Ruptured Aneurysms NIHSS of 0 or 1 13 (68.4%) 18(100%)  0.020 mRS score of 0-2 14 (73.7%) 17 (94.4%) 0.180 Patients with(N = 93) (N = 89) strokes < 10 cc's** NIHSS of 0 or 1 83 (89.3%) 85(95.5%) 0.164 mRS score of 0-2 87 (93.6%) 84 (96.6%) 0.499 UnrupturedAneurysm (N = 74) (N = 71) Patients with strokes < 10 cc's** NIHSS of 0or 1 70 (94.6%) 67 (94.4%) 1.000 mRS score of 0-2 73 (98.7%) 67 (97.1%)0.609

Neurocognitive Outcomes

Outcomes from the battery of neurocognitive tests are detailed inSupplementary Table 7. TAT-NR2B9C treated patients showed neurocognitivebenefits after treatment with TAT-NR2B9C, including significantimprovements in the neuropsychiatric inventory questionnaires.Surprisingly, although the numbers of patients were small forstatistical analysis in the ruptured aneurysm group, there weresignificant trends of improvement in cognitive and neuropsychiatrictests for patients treated with TAT-NR2B9C.

Safety

A total of 3 patients died. One, enrolled with an unruptured aneurysm,had received TAT-NR2B9C but died on day 3 due to hemorrhagic shock froma retroperitoneal hematoma induced by the groin puncture. Two receivedplacebo. One died on day 13 from neurological complications following anintra-procedural rupture of a previously unruptured aneurysm. The other,enrolled with a ruptured aneurysm, died on day 12 due to neurologicalcomplications of the SAH.

A listing of Adverse Events (AEs), including Severe and Serious AE's, isprovided in Table 5. Overall, the AE's were well balanced between thetreatment groups. There were a total of 388 AEs in Placebo, and 336 AEsin the TAT-NR2B9C group. However, only 2 AEs were deemed to be probablyrelated to drug and both were mild-consisting of transient hypotensionthat resolved within minutes. Of all Serious AEs (AEs that result indeath, are life-threatening, require a new hospitalization or prolongcurrent hospitalization, or result in a persistent or significantdisability or incapacity), 24 occurred in 14 patients who receivedplacebo, and 11 occurred in 9 patients who received TAT-NR2B9C (Table10). None were related to TAT-NR2B9C.

Another surprising finding is that patients receiving TAT-NR2B9C hadsignificantly fewer incidents of pain reported associated with theprocedure. Although the number of patients reporting pain associatedwith the aneurysm repair procedures was not high, the effect was largeenough that there is a P value of less than <0.02. Thus, TAT-NR2B9C canreduce procedural and post-procedural pain in humans, and is likely toreduce pain either without aneurysm repair procedures or in otherprocedures that have pain associated with them as a side effect. AsPSD-95 and the NMDAR2 subunits are also highly conserved in animals,PSD-95 inhibitors are likely to be effective in animals for pain aswell.

TABLE 10 Safety End Points and Adverse Events (AEs). Study Group PlaceboTAT-NR2B9C Odds P (N = 93) (N = 92) Ratio Value Most Common AdverseEvents (>10% in any Treatment Group) Total Subjects 85 (91%) 83 (90%)With 1 + AE Headache 37 (40%) 42 (46%) 0.4199 Nausea 27 (29%) 33 (36%)0.3206 Vomiting 8 (9%) 12 (13%) 0.3307 Procedural Pain 10 (11%) 2 (2%)0.0178 Hypotension 6 (6%) 9 (10%) 0.4066 Hypertension 7 (8%) 4 (4%)0.3606 AE relationship to Study Drug Total: 388 (100%) 336 (100%)Unrelated 271 (37.4%) 251 (34.7%) Unlikely related 96 (13.3%) 72 (9.9%)Possibly related 21 (2.9%) 11 (1.5%) Probably related 0 (0.0%) 2 (0.3%)Total Adverse Events Mild 208 (28.7%) 187 (25.8%) Moderate 159 (22.0%)121 (16.7%) Severe* 21 (2.9%) 28 (3.9%) Serious** Adverse Events by BodySystem Total 24 (14) 11 (9) Gastrointestinal 2 (2) 0 (0) AdministrationSite 0 (0) 1 (1) Procedural 2 (2) 1 (1) Complications Nervous System 14(9) 3 (3) Genitourinary 1 (1) 1 (1) Respiratory 2 (1) 2 (1) Vascular 3(3) 3 (3) Infectious 0 (0) 0 (0) Neoplastic 0 (0) 0 (0) Blood and 0 (0)0 (0) Lymphatic immune 0 (0) 0 (0) Endocrine 0 (0) 0 (0) Metabolic and 0(0) 0 (0) nutritional Psychiatric 0 (0) 0 (0) Eye 0 (0) 0 (0)Ear/Labyrinth 0 (0) 0 (0) Cardiac 0 (0) 0 (0) Hepatobiliary 0 (0) 0 (0)Skin 0 (0) 0 (0) Muskuloskeletal 0 (0) 0 (0) Renal 0 (0) 0 (0)congenital/familial/ 0 (0) 0 (0) genetic *Severe AE is defined as anincapacitating adverse events that precludes the performance of normalactivities **Serious AE is defined as an AE that results in death, islife-threatening, requires a new hospitalization or prolongs currenthospitalization, results in a persistent or significant disability orincapacity, or is a congenital or birth defect.

Discussion

This trial of TAT-NR2B9C in procedurally induced strokes showed benefitof TAT-NR2B9C in reducing the numbers and the median volumes of embolicstrokes in patients with ruptured and unruptured intracranial aneurysms.In the pre-specified subgroup analyses, the patients who benefitted themost were those who were enrolled with a SAH due to a ruptured aneurysm,where treatment with TAT-NR2B9C reduced the numbers of strokes by 64%and the volumes by about 80%. Additionally, this subgroup of patientsexhibited improved neurological outcomes by NIHSS, and trended toimproved neurological outcomes by mRS and improved cognitive outcomes.Despite the small size of this subgroup of subjects with rupturedaneurysms (37 patients), the size of the TAT-NR2B9C effect was so largeso as to produce statistically significant results. This is surprisinggiven that the benefit of TAT-NR2B9C treatment is thought to occurprimarily through the reduction of ischemia, and ischemia is rarely seenbefore day 5 in SAH.

The validity of the overall results of ENACT is supported by severalfeatures of the trial. ENACT was conducted in a double-blinded mannersuch that patients, investigators and those analyzing the trial resultswere unaware of treatment assignments. Demographic features that mayinfluence outcomes from strokes or SAH were balanced and showed nointeraction with the treatment effect.

Most subjects in ENACT (80%) had unruptured aneurysms, for which the 30dmortality and morbidity of endovascular repair, including neurologicaland cognitive deficits, is <10%. By contrast, those with a SAH are atgreater risk of sustaining impairments in neurological and/or cognitivefunction. Nonetheless, the trend to improvement in the small SAH patientsubgroup was surprising, as ENACT was designed to test theneuroprotection hypothesis primarily using MRI criteria, in subjects atrisk of mainly small embolic strokes, and in a relatively small patientpopulation. In patients with a SAH, neurological status on enrollmentwas good (median mRS and NIHSS of 0 in both placebo and TAT-NR2B9Cgroups). The deterioration in SAH patients untreated with TAT-NR2B9C at30 days post treatment suggests worsening of their clinical status aftertheir initial assessments. The worsening may result from such being moresusceptible to neurological or cognitive complications of endovascularcoiling, or because of SAH initiating a cascade ofclinically-deleterious events. In any event, subjects with SAH receivingTAT-NR2B9C have a significantly better clinical outcome than subjectswith SAH that do not.

Discussion of Cerebral Ischemia in SAH

Patients with a SAH are at risk of suffering various differentcomplications of their SAH. These complications may independently causeinjury to the brain and affect clinical outcome. Such complicationsinclude the original brain injury incurred by the sudden rise inintracranial pressure (ICP) as a result of the aneurysm rupture, as wellas more delayed complications such as hydrocephalus or cerebral ischemiadue to cerebral vasospasm.

Cerebral ischemia in SAH is the result of cerebral arterial vasospasm,and complicates the clinical course of approximately 30% of cases. Theincidence of clinically-relevant vasospasm in SAH is highest betweendays 5 and 12 after the SAH. However, this complication is quiteuncommon in the first three days after a SAH. A patient's ultimateclinical outcome after a SAH likely depends on the several factors,including demographic factors such as age and co-morbidities, theseverity of the SAH, and the various complications of the SAH such ashydrocephalus and vasospasm. Many lines of evidence demonstrate thatcerebral ischemia due to vasospasm is not the sole contributor to anadverse clinical outcome from SAH (MacDonald, 2007; Kaptain et al.,2000).

Given that TAT-NR2B9C was used in SAH subjects within 72 hours of theonset of the SAH, and given that cerebral ischemia in SAH is extremelyuncommon in the first three days after SAH, the beneficial effect ofTAT-NR2B9C observed in the ENACT trial in patients with a SAH isunlikely to be due to anti-ischemic effects, because TAT-NR2B9C is notbeing administered in a time-frame that can prevent cerebral ischemicdamage in SAH. The maximum plasma concentration (Cmax) occurs within 5minutes of the end of dose administration, and is mostly gone from theplasma in 30-45 minutes. The half-life of TAT-NR2B9C appears to be about5 hours. Therefore TAT-NR2B9C is unlikely to still be present in apatient's central nervous system at five days, the time at whichvasospasm occurrence becomes more common. Moreover, TAT-NR2B9C has beenshown not to alter cerebral blood flow (Bratane et al., Stroke. 2011November; 42(11):3265-70), and is therefore not expected to preventvasospasm in any way.

Thus it is unlikely that TAT-NR2B9C is improving neurological outcome inSAH patients by acting on cerebral ischemia because 1) it is notvasoactive and 2) it is not administered in the time frame when cerebralischemia is a complication of a SAH. Rather, TAT-NR2B9C may be improvingneurological outcome by addressing the primary brain injury that arisesafter a SAH before significant cerebral ischemia has developed. Itsefficacy may be due to a reduction of cell damage as a result of itsdisruption of cell signaling pathways that lead to the primary braininjury incurred by the rapid and transient rise in ICP that is known toarise after a SAH. Consequently, TAT-NR2B9C is addressing another factorof the various factors that may contribute to a patient's clinicaloutcome after a SAH, but not vasospasm or cerebral ischemia.

Although the invention has been described in detail for purposes ofclarity of understanding, it will be obvious that certain modificationsmay be practiced within the scope of the appended claims. Allpublications, accession numbers, and patent documents cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each were so individually denoted.To the extent more than one sequence is associated with an accessionnumber at different times, the sequences associated with the accessionnumber as of the effective filing date of this application is meant. Theeffective filing date is the date of the earliest priority applicationdisclosing the accession number in question. Unless otherwise apparentfrom the context any element, embodiment, step, feature or aspect of theinvention can be performed in combination with any other.

1. A method of treating a damaging effect of subarachnoid hemorrhage ina subject, comprising: administering an agent that inhibits binding ofPSD-95 to an NMDAR2 subunit to a subject having a subarachnoidhemorrhage, wherein the agent is a peptide linked to an internalizationpeptide, and the agent includes one or more D-amino acids, or the agentis a lipidated peptide, wherein development of neurocognitive deficitsin the subject is inhibited, and wherein the administering to thesubject is not in the course of a clinical trial.
 2. The method of claim1, wherein the subarachnoid hemorrhage is the result of physical trauma.3. The method of claim 1, wherein the subarachnoid hemorrhage occursspontaneously.
 4. The method of claim 3, wherein the subarachnoidhemorrhage is due to a ruptured aneurysm.
 5. The method of claim 3,wherein the subarachnoid hemorrhage is due to arteriovenousmalformation.
 6. (canceled)
 7. The method of any claim 1, wherein theagent inhibits development of infarctions detectable by MRI.
 8. Themethod of claim 1, wherein the subject receives endovascular surgery torepair a ruptured blood vessel causing the subarachnoid hemorrhage. 9.The method of claim 8, wherein the agent reduces pain resulting fromendovascular surgery.
 10. The method of claim 9, wherein the pain isalong a path traversed by an endoscope used in performing the endoscopicsurgery. 11-37. (canceled)
 38. The method of claim 1, wherein the agentis a peptide having an amino acid sequence consisting or comprising ofX1tSX2V (SEQ ID NO:7), wherein t and S are alternative amino acids, X1is selected from among E, Q, and A, X2 is selected from among A, Q, D,N, (N-Methyl)-A, (N-methyl)-Q, (N-methyl)-D, and (N-methyl) N.


39. The method or agent of claim 38, wherein the peptide is linked to aninternalization peptide.
 40. The method of claim 1, wherein the agent isa peptide having an amino acid sequence consisting or comprising ofYGRKKRRQRRRKLSSIESDV, SEQ ID NO:6 or YGRKKRRQRRRKLSSIETDV (SEQ IDNO:37), wherein one or more amino acids are D-amino acids.
 41. Themethod of claim 1 wherein the agent is a myristoylated peptide.
 42. Themethod or agent of claim 41, wherein peptide has an amino acid sequenceconsisting or comprising of KLSSIESDV or KLSSIETDV.
 43. The method ofclaim 42, wherein the myristoylation is at the N-terminus of thepeptide. 44-52. (canceled)